WO2020203961A1 - Lipid membrane structure and manufacturing method therefor - Google Patents

Lipid membrane structure and manufacturing method therefor Download PDF

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WO2020203961A1
WO2020203961A1 PCT/JP2020/014526 JP2020014526W WO2020203961A1 WO 2020203961 A1 WO2020203961 A1 WO 2020203961A1 JP 2020014526 W JP2020014526 W JP 2020014526W WO 2020203961 A1 WO2020203961 A1 WO 2020203961A1
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lipid
dispersion
coq
lipid membrane
membrane structure
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PCT/JP2020/014526
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French (fr)
Japanese (ja)
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勇磨 山田
原島　秀吉
光恵 日比野
悠介 佐藤
学 渡慶次
正寿 真栄城
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ルカ・サイエンス株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to a lipid membrane structure and a method for producing the same.
  • CoQ 10 is very lipophilic and cannot be applied to injection preparations, the main dosage form is tablets. However, in general, it is difficult to adapt tablets to the treatment of ischemic diseases that require prompt response, and despite the fact that CoQ 10 has functions such as strong antioxidant activity because the dosage form is tablets. The range of indications is narrowing. In response to this situation, the present inventors encapsulated CoQ 10 in lipid bilayer vesicles (liposomes) to obtain dispersibility in a solution, and further provided CoQ as a nanocapsule having a mitochondrial targeting ability. A 10- mounted MITO-Porter was constructed (Non-Patent Document 1, Patent Document 1).
  • CoQ 10 mounted formulations disclosed as MITO-Porter in these references includes a lipid membrane containing a dioleyl phosphatidyl ethanolamine and phosphatidic acid or sphingomyelin, lipid membrane structure containing octaarginine (R8) Become.
  • This system realizes efficient delivery of CoQ 10 to mitochondria, which is a source of active oxygen, and can suppress injury during ischemia.
  • Non-Patent Document 2 by intravenous administration to the liver ischemia model animals (mice), in the tissue of the animal, it was confirmed delivery and accumulation in the mitochondria into the cell CoQ 10 (Non-Patent Document 2 ).
  • Drug delivery system using nanocapsules such as liposomes allows existing drugs and nucleic acid drugs to be encapsulated or carried on the surface of particles, so that the drugs accumulate at the target site, increasing the therapeutic effect and reducing side effects. It brings about biological effects such as. Furthermore, nanocapsules are also applied to solubilization technology and contribute to improving the dispersibility of poorly water-soluble molecules such as CoQ 10 . It also protects the encapsulated drug from oxidation, can improve stability, and has a physicochemical effect.
  • Typical methods for preparing liposomes include a simple hydration method, a reverse phase evaporation method (REV) method, and an alcohol dilution method. It has been shown that when the above-mentioned CoQ 10- loaded MITO-Porter is prepared by an ethanol dilution method, particles having the highest CoQ 10 loading rate (drug content with respect to lipid) can be obtained (Non-Patent Document 3). ).
  • BCS class 4 Biopharmaceutics Classication System, Table 1
  • BCS class 4 Biopharmaceutics Classication System, Table 1
  • drugs belonging to BCS class 4 have poor gastrointestinal absorption due to poor gastrointestinal membrane permeability, and are generally considered to be difficult to formulate.
  • BCS class 4 drugs are poorly water-soluble, it is difficult to make them into injections, and because they are poorly permeable to the gastrointestinal membrane, it is difficult to tablet them as oral preparations to exert a high effect. is there.
  • CoQ 10 mounted MITO-Porter is the therapeutic effect can be expected.
  • the particle size of the CoQ 10- equipped MITO-Porter obtained repeatedly under the same conditions is in the range of 80 to 120 nm, and the particle size is uniform.
  • the CoQ 10- equipped MITO-Porter can be surface-modified with a polyarginine peptide typified by R8 (octaarginine), and the surface charge can be adjusted so as to have a predetermined zeta potential (for example, in the range of 15 to 25 mV). ..
  • a predetermined zeta potential for example, in the range of 15 to 25 mV.
  • the surface is modified with a polyarginine peptide after the preparation of the nanocapsules, but the reproducibility of the surface charge is poor and it is difficult to stably prepare the nanocapsules having a predetermined zeta potential.
  • the amount of CoQ 10- MITO-Porter prepared in one preparation was also small (for example, about 400 ⁇ L).
  • a new method capable of efficiently encapsulating, stable, and preparing nanocapsules having a smaller particle size than that obtained by the conventional method can be provided. Is desired.
  • MITO-Porter By including a poorly water-soluble compound, MITO-Porter can be efficiently solubilized and can be an efficient means of transport to mitochondria. If it is possible to efficiently encapsulate poorly water-soluble molecules other than CoQ 10 and to prepare nanocapsules that are stable and have a smaller particle size than that obtained by the conventional method, many water-resistant molecules belonging to BCS class 4 are poorly water-soluble. The application of sex compounds to pharmaceuticals can be greatly expanded.
  • lipid membrane structure or lipid
  • lipid that contains a poorly water-soluble compound such as that classified as BCS Class 4 containing CoQ 10 and has a smaller particle size and / or a smaller polydispersity index.
  • a method for producing a lipid film structure (or lipid nanoparticles) capable of preparing (nanoparticles) with good reproducibility and mass production is provided.
  • the present invention is as follows.
  • [1] A dispersion containing a poorly water-soluble compound and having a lipid film structure having an average particle size of 60 nm or less measured by a dynamic light scattering (DLS) method as a dispersoid in a dispersion medium, wherein the lipid film is contained.
  • [2] The dispersion according to [1], wherein the phospholipid is diorail phosphatidylethanolamine, phosphatidic acid and / or sphingomyelin.
  • a method for producing a dispersion which comprises a step of diluting the alcohol solution with an aqueous solvent and recovering a dispersion containing a lipid film structure containing a poorly water-soluble compound as a dispersoid from an outlet of a microchannel.
  • the phospholipid is dioleylphosphatidylethanolamine and phosphatidic acid and / or sphingomyelin
  • the membrane-permeable peptide is a polyarginine peptide consisting of 4 to 20 consecutive arginine residues.
  • the lipid membrane structure contains a phospholipid, a membrane-permeable peptide, and a lipid-modified polyethylene glycol.
  • the dilution flow path has a two-dimensionally bent flow path portion at least in a part thereof, and the bent flow path is formed.
  • the axial direction of the dilution flow path upstream from this or the extension direction thereof is the X direction
  • the width direction of the dilution flow path perpendicular to the X direction is the Y direction.
  • the structure that regulates the flow path width of the dilution flow path is formed at regular intervals d1, d2. .. .. It is a flow path structure formed by providing at least two of them, and the alcohol solution is introduced into the first introduction path, and the aqueous solvent is introduced into the second introduction path, [12] to [18]. ]
  • the manufacturing method according to any one of. [20] The dispersion according to any one of the above [1] to [11].
  • a lipid membrane structure comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleyl phosphatidylethanolamine, and lipid-modified polyethylene glycol.
  • a lipid membrane structure is a dispersion that contains precursors in the biosynthetic pathway between the inner and outer membranes of ubiquinone or its mitochondria. [21] The dispersion according to any one of the above [1] to [11].
  • a dispersion comprising a lipid membrane structure comprising lipid-modified polyethylene glycol, wherein the lipid membrane structure comprises curcumin.
  • the lipid membrane structure is a dispersion having an average particle size of 20 to 150 nm by the DLS method and a polydispersity index of 0.3 or less.
  • a dispersion in which the membrane-permeable peptide is a peptide selected from the group consisting of octaarginine (R8) and S2 peptides.
  • BCS Biopharmaceutics Classification System
  • lipid membrane structure has a polydispersity index (PDI) of 0.3 or less measured by a dynamic light scattering method.
  • PDI polydispersity index
  • the lipid membrane contains one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and dioleylphosphatidylethanolamine. Body or composition.
  • the lipid membrane structure further contains a membrane-permeable peptide and expresses the membrane-permeable peptide.
  • the membrane-permeable peptide can also coexist when producing the lipid nanoparticles, and the lipid nanoparticles containing the membrane-permeable peptide can be produced in one step, and as a result, the physical properties of the obtained lipid nanoparticles can be improved. Be controlled. Moreover, by modifying the surface of the lipid nanoparticles with the cationic polymer, the zeta potential of the lipid nanoparticles could be stably controlled.
  • lipid membrane structure containing a poorly water-soluble compound (eg, a BCS class 4 compound) having a more homogeneous particle size and / or a smaller particle size. Nanoparticles) can be prepared in large quantities.
  • the present invention it is possible to provide poorly water-soluble molecule-encapsulating lipid nanoparticles having a particle size of 60 nm or less, which has not been obtained in the past. Furthermore, the lipid nanoparticles have a smaller polydispersity index, and as a result, have the effect of increasing the efficiency of uptake into cells and mitochondria in combination with the small particle size.
  • a schematic explanatory diagram of a method for preparing a lipid membrane structure (or lipid nanoparticles or nanocapsules) using a microchannel device in an example is shown.
  • the experimental result (before dialysis) when the flow velocity ratio of the lipid phase and the aqueous phase was adjusted in Example 1 is shown.
  • the experimental result (after dialysis) when the flow velocity ratio of the lipid phase and the aqueous phase was adjusted in Example 1 is shown.
  • the result of the influence test of the dialysis temperature in Example 1 is shown.
  • the result of the stability test in Example 1 is shown. Comparative Example 1 shows the physical properties of the lipid nanoparticles obtained by the conventional method.
  • Example 3 The particle size, PdI and zeta potential of the lipid membrane structure (or lipid nanoparticles) obtained by the conventional method (Comparative Example 1) and Example 1 (before and after dialysis) in Example 2 are shown.
  • the cell uptake evaluation by flow cytometry (FACS) and the intracellular localization observation result by confocal laser scanning microscope (CLMS) using cervical cancer HeLa cells in Example 3 are shown.
  • the intracellular localization observation (HeLa cell) image in Example 3 is shown.
  • the intracellular localization observation (model disease cell) image in Example 3 is shown.
  • the intracellular localization observation (Human CDC cell) image in Example 3 is shown.
  • An image of intracellular localization observation (Human pulponary artery smooth muscle cells) in Example 3 is shown.
  • FIG. 5 shows a transmission electron microscope image of the CoQ 10- containing lipid membrane structure (or lipid nanoparticles) in Example 1 by negative staining.
  • OCR oxygen consumption rate
  • Example 7-1 Shows the scheme of therapy experiments with CoQ 10 containing lipid membrane structure of the liver injury model mice in Example 7-2 (or lipid nanoparticle). The results of the treatment experiment with the CoQ 10- containing lipid membrane structure (or lipid nanoparticles) of the liver disorder model mouse in Example 7-2 are shown. The average particle size and PdI of the curcumin-containing lipid membrane structure (or lipid nanoparticles) before and after dialysis in Example 8 are shown.
  • the dispersion is a composition containing a dispersoid and a dispersion medium.
  • the dispersoid is a lipid membrane structure containing a poorly water-soluble compound, and the dispersion medium can be an aqueous solvent.
  • the dispersion can preferably be a colored or uncolored clear solution.
  • the dispersoid is a lipid membrane structure (or lipid nanoparticles) containing a phospholipid and a lipid-modified uncharged hydrophilic polymer (eg, lipid-modified polyethylene glycol).
  • the lipid nanoparticles may preferably represent a membrane-permeable molecule (eg, a peptide) that promotes permeability to the cell membrane.
  • the lipid membrane structure has a lipid membrane structure containing a lipid-modified uncharged hydrophilic polymer in addition to phospholipids, whereby the dispersibility of the dispersoid in the dispersion is improved, and the dispersion (for example, a solution) ) Is prevented from becoming turbid.
  • the phospholipid the following phospholipids can be used, and preferably, one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and dioleylphosphatidylethanolamine can be contained.
  • the lipid membrane structure may further comprise the charged substances and / or membrane permeable molecules described below.
  • the uncharged hydrophilic polymer that can be used in the present invention can be a polymer that has no charge as a whole molecule and is hydrophilic.
  • the uncharged hydrophilic polymer is not particularly limited as long as it does not significantly inhibit the particle formation of the present invention, and examples thereof include polyoxazolines and polyalkylene glycols.
  • polyalkylene glycol polyethylene glycol can be preferably used.
  • polymer for example, a polymer having a number average molecular weight of 1,000 to 150,000 Da, preferably 1,000 to 5,000 Da (for example, about 2,000 Da) can be used.
  • the uncharged hydrophilic polymer can be linked to the lipid and complexed into a lipid membrane structure.
  • lipid membrane structure From the viewpoint of facilitating detachment from the lipid membrane structure, it can be linked to a lipid that is easily detached from the lipid membrane.
  • diacylglycerol for example, 1,2-dimyristoyl-sn-glycerol
  • Lipid-modified uncharged hydrophilic polymers can be added to prevent turbidity in the dispersion.
  • the lipid membrane structure is obtained as nanoparticles by mixing a lipid phase in which lipids are dissolved and an aqueous phase on a microchannel device containing a baffle mixer. Details of the manufacturing method will be described later.
  • an organic solvent for example, alcohol can be used as the solvent.
  • alcohols for example, t-butanol, 1-propanol, 2-propanol and 2-butoxyethanol can be used as long as biotoxicity is not significantly caused, and ethanol can be preferably used.
  • the poorly water-soluble compound can be made soluble in the alcohol. If heating is required for dissolution, the poorly water-soluble compound may be dissolved in alcohol by heating. Alcohol may be selected from the viewpoint of increasing solubility depending on the type of the poorly water-soluble compound to be included.
  • Lipid membrane structures include phospholipids and lipid-modified uncharged hydrophilic polymers (eg, lipid-modified polyethylene glycol).
  • the dispersoid has a particle size measured by the DLS method of 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, preferably 55 nm or less, or more preferably 50 nm.
  • the lipid membrane structure may have a particle size of 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, or 50 nm or more in the particle size measured by the DLS method.
  • the number average particle size of the lipid membrane structure can be, for example, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, or 50 nm or more, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, It can be 110 nm or less, 100 nm or less, 95 nm or less, 90 nm or less, 85 nm or less, 80 nm or less, 75 nm or less, 70 nm or less, 65 nm or less, or 60 nm or less. In some embodiments, the number average particle size can be 30 nm to 150 nm or less, for example 50 nm to 100 nm. As described above, the particle size may be the particle size obtained by the DLS method.
  • the lipid membrane structure (or lipid nanoparticles) has a polydispersity index (PDI) obtained by the DLS method of 0.5 or less, 0.45 or less, 0.4 or less, 0.35 or less, 0.3 or less. , 0.25 or less, or 0.2 or less.
  • PDI means that 1 is the maximum, and the smaller the value, the more uniform the particle size (the particle size distribution becomes sharper and / or monodisperse).
  • the PDI of the lipid membrane structure (or lipid nanoparticles) is 0.3 or less.
  • the lipid membrane structure (or lipid nanoparticles) is The particle size measured by the DLS method is 30 nm to 150 nm. It contains a lipid membrane structure (or lipid nanoparticles) having a PDI determined by the DLS method of 0.3 or less.
  • the particle size is, for example, 30 nm to 40 nm, 40 nm to 150 nm, 40 nm to 140 nm, 40 nm to 130 nm, 40 nm to 120 nm, 40 nm to 110 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm.
  • the lipid membrane structure (or lipid nanoparticles) is The particle size measured by the DLS method is 30 nm to 150 nm. It contains 50% or more, 60% or more, 70% or more, or 80% or more of lipid membrane structures (or lipid nanoparticles) having a PDI determined by the DLS method of 0.3 or less.
  • the particle size is, for example, 30 nm to 40 nm, 40 nm to 150 nm, 40 nm to 140 nm, 40 nm to 130 nm, 40 nm to 120 nm, 40 nm to 110 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm.
  • the lipid membrane structure (or lipid nanoparticles) is The number average (number average particle size) of the particle size measured by the DLS method is 30 nm to 150 nm. It contains a lipid membrane structure (or lipid nanoparticles) having a PDI determined by the DLS method of 0.3 or less.
  • the number average particle size is, for example, 30 nm to 40 nm, 40 nm to 150 nm, 40 nm to 140 nm, 40 nm to 130 nm, 40 nm to 120 nm, 40 nm to 110 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm.
  • the lipid membrane structure (or lipid nanoparticles) comprises phospholipids and lipid-modified polyethylene glycol.
  • the lipid membrane structure is a cationic polymer such as a cell membrane penetrating peptide (eg, a polymer of cationic amino acids, eg, polyarginine).
  • the polymer may be expressed.
  • a conjugate of the cationic polymer and a lipid for example, a myristol group
  • the lipid membrane is formed on the lipid portion of the conjugate.
  • the membrane-permeable peptides include tat peptide (having a peptide sequence corresponding to the 48 to 60th position of the amino acid sequence of AAF35362.1, GenBank registration number of tat protein of human immunodeficiency virus), oligoarginine (R9), oligolysine.
  • tat peptide having a peptide sequence corresponding to the 48 to 60th position of the amino acid sequence of AAF35362.1, GenBank registration number of tat protein of human immunodeficiency virus
  • R9 oligoarginine
  • oligolysine examples thereof include peptides rich in basic amino acids such as (K10), amphipathic peptides having a basic portion and a hydrophobic portion such as penetratin, and peptides such as transportin and TP10.
  • Membrane-permeable peptides having mitochondrial orientation include lipophilic cations such as octaarginine (R8), lipophilic triphenylphosphonium cation (TPP) or rhodamine 123, and mitochondria.
  • Target sequence Mitochondrial Targeting Sequence; MTS
  • MTS Mitochondrial Targeting Sequence
  • S2 peptide Szeto, H. R. 2011, 28, pp. 2669-2679
  • the S2 peptide Dmt-D-Arg-FK-Dmt-D-Arg-FK-NH 2 ⁇ Here, Dmt is 2,6-dimethyltyrosine, and D-Arg is D-form arginine. Yes, F is L-form phenylalanine and K is L-form lysine ⁇ .
  • the zeta potential of the lipid membrane structure can be 5 mV or higher, 10 mV or higher, 15 mV or higher, 16 mV or higher, 17 mV or higher, 18 mV or higher, 19 mV or higher, or 20 mV or higher, for example, about 20 mV. ..
  • the dispersions of the invention contain less than 1%, less than 0.5%, less than 0.1%, or less than 0.05% alcohol that can be used during production in the dispersion medium. Or does not contain alcohol.
  • the dispersion can be dialyzed with an alcohol-free external dialysis solution.
  • the external dialysis solution can be, for example, an aqueous solution such as physiological saline.
  • the dispersion of the present invention comprises a lipid membrane structure containing a poorly water soluble compound (eg, a BCS class 4 compound such as curcumin and / or CoQ 10 ).
  • a poorly water soluble compound eg, a BCS class 4 compound such as curcumin and / or CoQ 10
  • the poorly water-soluble compound can be a compound exhibiting solubility in ethanol.
  • the lipid membrane structure contains 10-40 mol% (preferably 20-40 mol%) of CoQ 10 relative to the total amount of lipid membrane, eg 20. Included in the range of ⁇ 30 mol%).
  • the lipid membrane structure containing CoQ 10 may use ubiquinone (oxidized ubiquinone) instead of CoQ 10 , or may use a precursor of ubiquinone or CoQ 10 in the biosynthetic pathway between the inner and outer membranes of mitochondria. May be good.
  • Ubiquinone is called CoQ n , corresponding to the number n of repeating units of the isoprene.
  • n can be a natural number in the range of 4 to 15 or 6 to 12, for example, a natural number in the range of 6 to 10, for example, in the range of 8 to 12. It can be a natural number, eg, a natural number in the range 8-10, eg, 10.
  • Examples of these precursors include dimethoxyubiquinone (DMQ) and 5-hydroxyubiquinone (5-HQ).
  • DMQ dimethoxyubiquinone
  • 5-HQ 5-hydroxyubiquinone
  • the R8-expressing lipid membrane structure of the present invention was considered to have delivered ubiquinone to the inner mitochondrial membrane, and was able to improve the oxygen consumption rate of cells. Therefore, the R8 expressive lipid membrane structure of the present invention can be used to deliver ubiquinone from extracellular to the inner mitochondrial membrane.
  • the dispersion comprises one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleylphosphatidylethanolamine, and steallylated polyethylene glycol.
  • the lipid membrane structure comprises precursors (preferably CoQ n , particularly CoQ 10 ) in the biosynthetic pathway between the inner and outer membranes of ubiquinone or its mitochondria.
  • the lipid membrane structure includes a lipid membrane structure having a particle size of 20 to 100 nm according to the DLS method, and has a PDI of less than 0.3.
  • the dispersion (composition) in this preferred embodiment can have a number average particle size of 50 nm to 70 nm.
  • the dispersion medium can be saline, preferably less than 1%, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0 alcohol. .Contains less than 1%, or less than 0.01%, or less than the detection limit, or does not contain alcohol.
  • the lipid membrane structure may further express a cell membrane penetrating peptide (eg, R8). The lipid membrane structure is non-hollow.
  • the dispersion comprises one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleylphosphatidylethanolamine, and steallylated polyethylene glycol.
  • the lipid membrane structure comprises curcumin, the lipid membrane structure comprises a lipid membrane structure having a particle size of 40-300 nm according to the DLS method, and the PDI is less than 0.3. ..
  • the dispersion (composition) in this preferred embodiment can have a number average particle size of 100 nm to 120 nm.
  • the dispersion medium can be saline, preferably less than 1%, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0 alcohol. .Contains less than 1%, or less than 0.01%, or less than the detection limit, or does not contain alcohol.
  • the lipid membrane structure may further express the cell membrane penetrating peptide and / or mitochondrial directional peptide (eg, R8 and / or S2 peptide).
  • the method for producing a dispersion of the present invention can be used to produce the above-mentioned dispersion.
  • an alcohol solution in which a phospholipid, a membrane-permeable peptide, a lipid-modified polyethylene glycol, and a poorly water-soluble compound are dissolved and an aqueous solvent are continuously connected to the entrance of the microchannel of the microchannel structure.
  • the alcohol solution is mixed (that is, diluted) with an aqueous solvent in the microchannel, and a dispersion containing a lipid membrane structure containing a poorly water-soluble compound as a dispersoid is provided in the microchannel.
  • the lipid membrane structure (dispersant) thus obtained is lipid nanoparticles.
  • the nanoparticles are non-hollow structures filled with phospholipids or lipid membranes (non-hollow structures; or non-hollow lipid nanoparticles; or non-hollow if they represent mitochondrial directional molecules). It can be a hollow MITO-Porter).
  • the dispersoid in the present invention may be referred to as lipid nanoparticles (or nanocapsules).
  • the phospholipid is not particularly limited, and is, for example, phosphatidylcholine (for example, dioleoil phosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.) Phosphatidylglycerol, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.
  • phosphatidylcholine for example, dioleoil phosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatid
  • phosphatidylserine phosphatidylinositol, phosphatidylic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramidephosphorylglycerol, ceramidephosphorylglycerol phosphate, 1,2-dipalmitoyl-1,2- It can be deoxyphosphatidylcholine, plasmalogen, egg yolk lecithin, soybean lecithin, hydrogenated additives thereof and the like.
  • Phospholipids are preferably phosphatidylethanolamine (eg, diolaylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, etc.) and sphingomyelin. More preferably, diorail phosphatidylethanolamine and sphingomyelin.
  • phosphatidylethanolamine eg, diolaylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, etc.
  • sphingomyelin More preferably, diorail phosphatidylethanolamine and sphingomyelin.
  • a lipid membrane containing a phospholipid can contain a charged substance in addition to the phospholipid, and the charged substance is a component of the lipid membrane capable of imparting a positive charge or a negative charge to the lipid membrane, and is a lipid.
  • the amount of charged substance contained in the membrane is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, and more preferably 20% (molar ratio) or less of the total amount of substances constituting the lipid membrane. ..
  • the lower limit of the content of the charged substance is 0.
  • Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane, and the like, and negative charges are given.
  • Examples of the charged substance to be imparted include disetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidyl inositol, phosphatidyl acid and the like.
  • the zeta potential of the lipid membrane structure can be 5 mV or higher, 10 mV or higher, 15 mV or higher, 16 mV or higher, 17 mV or higher, 18 mV or higher, 19 mV or higher, or 20 mV or higher, for example, about 20 mV. ..
  • the phospholipids are diorail phosphatidylethanolamine and phosphatidic acid and / or sphingomyelin (ie, one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and diorail phosphatidylethanolamine. Included) is preferred for effective delivery of the poorly water-soluble compound, which is the substance of interest, into the mitochondria.
  • the membrane permeable molecule can be, for example, a cationic polymer.
  • the membrane permeable molecule can be, for example, a membrane permeable peptide.
  • Membrane-permeable peptides are membrane-permeable domains that are effective for the effective delivery of poorly water-soluble compounds of interest into mitochondria.
  • the membrane-permeable peptide can be the membrane-permeable peptide described in paragraphs 0052 to 0092 of Patent Document 1, preferably a polyarginine peptide consisting of 4 to 20 consecutive arginine residues.
  • the polyarginine peptide preferably consists of 6-12, more preferably 7-10 contiguous arginine residues, or 8 contiguous arginine residues.
  • Membrane-permeable peptides can be linked to lipids. Thereby, the membrane-permeable peptide can be contained in the lipid membrane structure, and the membrane-permeable peptide can be expressed on the lipid membrane structure.
  • the zeta potential of the lipid membrane structure can be 5 mV or higher, 10 mV or higher, 15 mV or higher, 16 mV or higher, 17 mV or higher, 18 mV or higher, 19 mV or higher, or 20 mV or higher, for example, about 20 mV. ..
  • Lipid-modified polyethylene glycol is a component that imparts hydrophilicity to lipid membranes.
  • Lipid-modified polyethylene glycol (PEG) is a compound obtained by lipid-modifying polyethylene glycol (PEG), and the molecular weight of polyethylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably 1. It is about 000 to 5,000.
  • the molecular weight of PEG is represented by a number average molecular weight.
  • Examples of the lipid-modified polyethylene glycol can be dioleoylglycerol-modified PEG, dilauroylglycerol-modified PEG, dimyristoylglycerol-modified PEG, dipalmitoylglycerol-modified PEG, distearoylglycerol-modified PEG and the like. More specifically, as the lipid-modified polyethylene glycol (lipid-modified PEG), stearyllated polyethylene glycol (for example, PEG45 stearate (STR-PEG45)) can be used.
  • stearyllated polyethylene glycol for example, PEG45 stearate (STR-PEG45)
  • a polyethylene glycol derivative such as amine can also be used. However, it is not limited to these.
  • the poorly water-soluble compound can be, for example, a compound belonging to Biopharmaceutics Classification System (BCS) class 4 without limitation.
  • Poorly water-soluble compound is not particularly limited, for example, terfenadine (Terfenadine), furosemide (furosemide), cyclosporine (Cyclosporin), acetazolamide (acetazolamide), colistin (Colistin), mebendazole (Mebendazole), coenzyme Q 10 (CoQ 10) And so on.
  • the alcohol in the alcohol solution is not particularly limited, and examples thereof include ethanol, t-butanol, 1-propanol, 2-propanol, and 2-butoxyethanol.
  • the concentration of each component of the alcohol solution can be appropriately determined according to the desired lipid membrane structure (or lipid nanoparticles), and is not particularly limited.
  • Phospholipids are, for example, in the range of 50-80 mol%
  • Membrane-permeable peptides are, for example, in the range of 5-20 mol%
  • Lipid-modified polyethylene glycols are, for example, in the range of 1-10 mol%.
  • the poorly water-soluble compound can be in the range of 10 to 40 mol%, for example.
  • aqueous solvent examples include an aqueous solution, for example, water or basically water as a main component, for example, a physiological saline solution, a buffer aqueous solution (for example, a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, etc.). ), Etc., which can be preferably used in the present invention.
  • phosphate buffer may be preferably used.
  • an alcohol solution containing a phospholipid or the like is diluted with an aqueous solvent, and a dispersion containing a lipid membrane structure containing a poorly water-soluble compound as a dispersoid is provided as a microchannel. Collect from the exit of.
  • the microchannel structure used in the method for producing a dispersion of the present invention is prepared by diluting an alcohol solution containing a phospholipid or the like with an aqueous solvent to prepare a dispersion containing a lipid membrane structure as a dispersoid.
  • Any microchannel structure that can be used can be used without particular limitation. Such a microchannel structure is described in, for example, Patent No.
  • Patent Document 2 Japanese Patent No. 6234971, Non-Patent Document 4, Non-Patent Document 5, WO2018 / 190423 A1 (Patent Document 2) and the like. Can be exemplified.
  • a lipid membrane having a uniform particle size with a small degree of dispersion while controlling the particle size of the lipid membrane structure to a desired value. It can be advantageously used to obtain a dispersion containing the structure as a dispersoid.
  • the microchannel structure described in Patent Document 2 has a first introduction path for introducing a first fluid and a second introduction path for introducing a second fluid, which are independent of each other on the upstream side.
  • Each of which has a constant length and merges to form one dilution channel toward the downstream side thereof, and the dilution channel is bent (for example, two-dimensionally bent) at least in a part thereof.
  • the bent flow path portion has a flow path portion, and the axial direction of the dilution flow path upstream from this or the extension direction thereof is the X direction, and the width direction of the dilution flow path perpendicular to the X direction is Y.
  • the direction is approximately Y (approximately + Y) toward the center of the flow path alternately from both side walls of the dilution flow paths facing each other in the Y direction.
  • And has a constant width x1 and x2 in the X direction. .. ..
  • the structure that regulates the flow path width of the dilution flow path is formed at regular intervals d1, d2. .. .. It is a flow path structure formed by providing at least two of them.
  • the flow path width y0 is preferably 20 to 1000 ⁇ m, more preferably 100 to 400 ⁇ m, and further preferably 150 to 300 ⁇ m.
  • the heights of each structure h1, h2. .. .. Is preferably 1 / 2y0 or more and 3/4y0 or less.
  • the structure is preferably provided in the range of 10 to 100, more preferably 10 to 50, and even more preferably 15 to 30.
  • an alcohol solution is introduced into the first introduction path as a lipid phase, and an aqueous solvent is introduced into the second introduction path as an aqueous phase.
  • the distance from the confluence of the first introduction path and the second introduction path to the upstream end of the first structure is appropriately set, but the diluted fluid at the set speed flowing between them is 0.1 seconds or less. It is preferable that it is specified according to the set speed of the diluting fluid so as to pass through.
  • the flow path may be heated.
  • the amount of each supply of the alcohol solution and the aqueous solvent to the microchannel is controlled so that the alcohol concentration of the dispersion recovered from the outlet of the microchannel is 40% or less, which is the desired particle size and dispersion degree. It is preferable because it is possible to obtain a dispersion containing the lipid film structure having the above as a dispersoid.
  • the alcohol concentration of the dispersion recovered from the outlet of the microchannel is preferably controlled to be in the range of 5 to 35%, more preferably in the range of 10 to 30%.
  • the operation in the microchannel structure can be carried out at a temperature in the range of 0 to 70 ° C., for example, while considering the boiling point of the solvent.
  • the alcohol solution is diluted with an aqueous solvent in the microchannel, and the dispersion containing the lipid membrane structure containing the poorly water-soluble compound as a dispersoid is recovered from the outlet of the microchannel.
  • a step of removing alcohol from the dispersion recovered from the outlet of the microchannel can be further included.
  • Alcohol removal from the dispersion can be performed, for example, by dialysis, distillation, or the like. Dialysis can be performed, for example, from 0 ° C. to room temperature.
  • the dispersion or the dispersion from which the alcohol has been removed can be further subjected to a concentration step.
  • the concentration step can be, for example, ultrafiltration, centrifugation, evaporation of solvent (water) or dialysis.
  • Ultrafiltration can be performed using, for example, an ultrafiltration membrane.
  • the ultrafiltration membrane has a predetermined nominal molecular weight cutoff (NMCO), and the predetermined NMCO can be any NMCO in the range of 50 kDa to 200 kDa, and is included in the dispersion to be obtained. It can be appropriately selected according to the particle size of the lipid membrane structure.
  • the present invention is a dispersion containing a poorly water-soluble compound and containing a lipid film structure having an average particle diameter of 60 nm or less measured by a dynamic light scattering (DLS) method as a dispersant in a dispersion medium.
  • the lipid membrane of the lipid membrane structure is the dispersion containing phospholipids and lipid-modified polyethylene glycol.
  • the lipid membrane of the lipid membrane structure can contain a membrane-permeable peptide.
  • the membrane-permeable peptide is not contained as an alcohol solution (lipid phase introduced into the microchannel) containing phospholipids and the like. Use an alcohol solution.
  • Phospholipids are preferably dioleylphosphatidylethanolamine and phosphatidic acid and / or sphingomyelin for effective delivery of the poorly water-soluble compound which is the target substance into the mitochondria.
  • the content of the membrane-permeable peptide is, for example, 5 to 20 mol%, preferably 10 to 15%, based on the total amount of the lipid membrane. It is in the range of mol%.
  • the content of lipid-modified PEG in the lipid membrane structure (or lipid nanoparticles) is, for example, in the range of 1 to 10 mol%, preferably 3 to 5 mol%, based on the total amount of the lipid membrane.
  • the amount of the poorly water-soluble compound in the lipid membrane structure (or lipid nanoparticles) is, for example, in the range of 10 to 40 mol%, preferably 15 to 30 mol%, or 20 to 25 mol%, based on the total amount of the lipid membrane. Is the range of.
  • the polydispersity index (PDI or PdI) of the lipid membrane structure (or lipid nanoparticles) measured by the DLS method is preferably 0.3 or less, preferably 0.25 or less.
  • the zeta potential of the lipid membrane structure is not particularly limited, but is, for example, in the range of 10 mV or more, 11 mV or more, 12 mV or more, 13 mV or more, 14 mV or more, or preferably 15 mV or more. Is.
  • the zeta potential of the lipid membrane structure (or lipid nanoparticles) of the dispersion of the present invention can be measured by the Zetasizer Nano ZS (Malvern, Worcestershire, UK) using laser Doppler electrophoresis.
  • the dispersion medium of the dispersion of the present invention is an aqueous solvent.
  • aqueous solvents are as described above.
  • the dispersion medium does not contain alcohol, or alcohol is removed from the dispersion medium.
  • the dispersion of the present invention is used to transfer a poorly water-soluble compound to intracellular and intracellular mitochondria.
  • the dispersion of the present invention is not particularly limited, but is, for example, a dispersion in which the phospholipids are dioleylphosphatidylethanolamine, phosphatidylic acid and / or sphingomyelin, and CoQ 10 as a poorly water-soluble compound is used as the total amount of the lipid membrane.
  • lipid nanoparticles as a dispersoid.
  • the dispersion of the present invention comprises a lipid membrane structure (or lipid nanoparticles) comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin and dioleylphosphatidylethanolamine.
  • CoQ 10 is contained in the range of 10 to 40 mol% with respect to the total amount of the lipid membrane, and the lipid membrane structure having a particle size measured by the DLS method of 60 nm or less, preferably 55 nm or less, more preferably 50 nm or less ( Or lipid nanoparticles) and may further have a PDI of less than 0.3 when measured by the DLS method.
  • the lipid membrane structure (or lipid nanoparticles) is a dispersoid.
  • the average particle size is preferably small to some extent from the viewpoint of efficiency of uptake into cells, and is not particularly limited, but the lower limit value can be about 10 nm, preferably about 20 nm.
  • the dispersion of the present invention comprises a lipid membrane structure (or lipid nanoparticles) comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin and dioleylphosphatidylethanolamine.
  • the lipid membrane structure (or lipid nanoparticles) contained CoQ 10 in the range of 10 to 40 mol% with respect to the total amount of the lipid membrane, and the lipid membrane structure (or lipid nanoparticles) was measured by the DLS method. It can have an average particle size of 50 nm to 60 nm and a PDI of less than 0.3.
  • the lipid membrane structure (or lipid nanoparticles) is a dispersoid.
  • the poorly water-soluble compound can be a fat-soluble compound.
  • the poorly water soluble compound is soluble in alcohol (eg, ethanol).
  • the poorly water-soluble compound can be a fat-soluble BCS class 4 compound.
  • the poorly water soluble compound can be a BCS class 4 compound that is soluble in ethanol. In order to improve or improve the solubility of the poorly water-soluble compound in alcohol, it may further include dissolving the compound in alcohol under heating conditions.
  • CoQ 10 can be dissolved in ethanol under heating conditions (eg, 50 ° C.) for use in the preparation of lipid membrane structures (or lipid nanoparticles), and in the dissolved state the lipid membrane structures.
  • Heating conditions eg, 50 ° C.
  • it can be incorporated into the membrane in a state close to nature, and therefore, the original functionality is fully exhibited at the place where CoQ 10 delivered intracellularly is delivered. Can be.
  • a pharmaceutical preparation containing a dispersion containing the poorly water-soluble compound of the present invention is provided.
  • a pharmaceutical preparation containing the dispersion of the present invention containing CoQ 10 is provided.
  • the composition in which the poorly water-soluble compound is CoQ 10 can be administered to, for example, a subject having a dysfunction in the respiratory chain complex I.
  • the composition in which the poorly water-soluble compound is CoQ 10 is a subject having a dysfunction in the respiratory chain complex (for example, any one or more of the respiratory chain complexes I, II, III, and IV). It can be used to improve ATP production in cells in subjects suffering from mitochondrial disease.
  • Subjects having dysfunction in respiratory chain complex I are not particularly limited, but are, for example, subjects suffering from mitochondrial encephalomyopathy (MELAS), subjects suffering from Leigh encephalopathy, and subjects suffering from Leber's hereditary optic neuropathy (LHON). Can be mentioned.
  • MELAS mitochondrial encephalomyopathy
  • LHON Leber's hereditary optic neuropathy
  • the composition in which the poorly water-soluble compound is CoQ 10 includes, for example, a subject suffering from CoQ 10 deficiency.
  • the subject can be an animal, particularly a mammal, particularly a primate, particularly preferably a human.
  • a method for administering a poorly water-soluble compound to a subject which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention.
  • a method for delivering a poorly water-soluble compound into cells in a subject's body which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention. ..
  • a method for delivering a poorly water-soluble compound to mitochondria in a cell in a subject body which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention. Will be done.
  • a method for treating mitochondrial disease in a subject suffering from mitochondrial disease which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention.
  • a method for treating mitochondrial disease in a subject suffering from mitochondrial disease which comprises administering the dispersion of the present invention containing CoQ 10 to the subject.
  • the use of a poorly water-soluble compound in the production of a pharmaceutical preparation containing the dispersion of the present invention containing the poorly water-soluble compound is provided.
  • the use of CoQ 10 is provided in the manufacture of a pharmaceutical formulation comprising a dispersion dispersion of the present invention containing CoQ 10.
  • the present invention provides the use of phospholipids and lipid-modified uncharged hydrophilic polymers in the manufacture of pharmaceutical formulations comprising the dispersions of the present invention containing poorly water soluble compounds.
  • the present invention provides the use of phospholipids and lipid-modified uncharged hydrophilic polymers in the manufacture of pharmaceutical formulations comprising the dispersion dispersions of the present invention containing CoQ 10 .
  • INDUSTRIAL APPLICABILITY The present invention provides the use of poorly water-soluble compounds, phospholipids and lipid-modified uncharged hydrophilic polymers in the manufacture of pharmaceutical formulations comprising the dispersions of the present invention containing poorly water-soluble compounds.
  • the present invention in the manufacture of a pharmaceutical formulation comprising a dispersion dispersion of the present invention containing CoQ 10, CoQ 10, the use of phospholipids and lipid-modified uncharged hydrophilic polymer.
  • the dispersion of the present invention can be made into a pharmaceutical formulation.
  • Pharmaceutical formulations containing the dispersions of the invention may further contain pharmaceutically acceptable excipients.
  • Excipients include, but are not limited to, buffering agents, tonicity agents, pharmaceutically acceptable salts, dispersants, antioxidants, preservatives, and soothing agents.
  • the dispersion of the present invention can be prepared as a cosmetic or supplement. Therefore, according to the present invention, cosmetics and supplements containing the dispersion of the present invention may be provided.
  • 2. Make 500 mL of PBS (-) for dialysis and store at 25 ° C. with stirring.
  • 3. Put 300-400 mL of DDW in a beaker, cut the dialysis membrane Spectra / Por 4 dialysis membrane (MWCO 12k-14k) to an appropriate length, stir vigorously so that the membrane does not stick, and hydrate (30 minutes or more). ).
  • 4. Add the following amounts of each solution to the Eppendorf tube to prepare a lipid solution (lipid phase).
  • Example 1 1-1 Preparation method of CoQ 10- MITO-Porter using microchannel
  • the particles were prepared using the microchannel device shown in FIG.
  • As the syringe pump a Standard Infusion Only Pump 11 Elite manufactured by HARVARD APPARATUS was used.
  • As the syringe 1 mL (lipid phase) and 2.5 mL (aqueous phase) of a glass syringe manufactured by HAMILTON were used.
  • (I) Lipid phase An ethanol solution containing 7.5 mM DOPE (EtOH), 7.5 mM SM (EtOH), and 7.5 mM DMG-PEG 2000 (EtOH) was prepared and brought to room temperature. An ethanol suspension containing a lipid material was prepared in an Eppendorf tube so as to have the volume ratio shown in the above figure, and was used as (i) a lipid phase. Further, the lipid phase contained 12.6 ⁇ L of 20 mg / mL STR-R8 so as to be modified by 10 mol% with respect to the lipid concentration.
  • (Iv) Flow Velocity Ratio The flow velocity ratio was adjusted so that the ethanol dilution concentration was 10%, 20%, 30%, or 40% in the lipid phase and the aqueous phase.
  • the flow rates of the lipid phase and the aqueous phase are 50 ⁇ L / min and 450 ⁇ L / min, 100 ⁇ L / min and 400 ⁇ L / min, 150 ⁇ L / min and 350 ⁇ L / min, or 200 ⁇ L / min and 300 ⁇ L / min, respectively. did.
  • Particles were prepared under each of the above flow rate ratio conditions (Fig. 2). As shown in FIG. 2, the particle size decreased as the ethanol dilution concentration decreased. Further, as shown in FIG.
  • the CoQ 10- MITO-Porter solution prepared in the microchannel was dialyzed for 2 hours (Fig. 1).
  • dialysis clips Spectra / Por Closures and dialysis membrane Spectra / Por 4 dialysis membrane (MWCO 12k-14k) manufactured by Spectram Laboratories were used.
  • the lipid nanoparticle solution tended to have a smaller particle size and an increased PdI due to dialysis as compared with before dialysis (FIG. 3).
  • DDS targeting the liver it has been shown that lipid nanoparticles having a particle size of 100 nm or less enable efficient delivery of the target substance, and that the smaller the particle size, the higher the efficiency.
  • the particles were used in the following examples. Neither the preparation of a dispersion having such a small particle size nor the preparation of a dispersion having such a small PdI was possible by the conventional method.
  • Stability Test In order to evaluate the stability of the prepared particles, a stability test was conducted at 4 ° C. and 25 ° C. under shading for 14 days (Fig. 5). During the storage period, the particle size increased over time at 25 ° C. On the other hand, at 4 ° C., the particle size was maintained at around 50 nm and was stable.
  • Comparative Example 1 Preparation method of CoQ 10- MITO-Porter using the conventional method Particles were prepared using the ethanol dilution method (Non-Patent Document 2). Prepare 7.5 mM DOPE (EtOH), 7.5 mM SM (EtOH), and 7.5 mM DMG-PEG 2000 (EtOH) and bring to room temperature. An ethanol suspension containing a lipid material was prepared in an Eppendorf tube so as to have the volume ratio shown on the right. Since CoQ 10 is in a suspended state and precipitation proceeds, ultrasonic treatment was performed immediately before use. PBS (-) buffer was added to the suspension and diluted to an ethanol concentration of 90%.
  • the CoQ 10 concentration and the CoQ 10 recovery rate contained in the prepared CoQ 10- MITO-Porter solution (conventional method) were quantified using HPLC and calculated from the calibration curve.
  • HPLC Agilent 1200 series was used.
  • the CoQ 10- MITO-Porter obtained using the microchannel was negatively stained and observed using a transmission electron microscope. The results were as shown in FIG. As shown in FIG. 16, the obtained CoQ 10- MITO-Porter contained a structure considered to be a lipid membrane inside the particles. As described above, it was clear that the CoQ 10- MITO-Porter obtained by using the microchannel was a non-hollow lipid structure.
  • CoQ 10 is poorly water-soluble, it is considered that CoQ 10 was incorporated into the lipid membrane, and the non-hollow lipid structure whose inside is filled with the lipid membrane is suitable for encapsulation of a large amount of CoQ 10. Was thought to be.
  • Example 2 2-1 Accuracy of particle preparation using microchannel
  • the results of Example 1 and Comparative Example 1 were compared.
  • the particle size tended to be small
  • the obtained dispersion was able to maintain uniform and highly accurate particles.
  • the obtained dispersion was about +20 mV even at the (C) zeta potential, and it is considered that there was no variation between preparations and that the dispersion could be modified with STR-R8 almost constantly.
  • the dispersion was modified with STR-R8 after the dispersion was formed, and it was difficult to reproducibly prepare the zeta potential at about +20 mV. Furthermore, the (D) CV value was used as an accuracy parameter for particle preparation.
  • the particle size CV of the dispersion was 0.082 in Comparative Example 1, whereas the particle size CV of the dispersion before and after dialysis of the method of the present invention was 0.062 and 0.065, respectively. there were.
  • the CV of the PdI of the dispersion was 0.192 in Comparative Example 1, whereas the CV of the PdI of the dispersion before and after dialysis of the method of the present invention was 0.148 and 0.066, respectively. ..
  • the CV of the zeta potential of the obtained dispersion was 0.171 in the conventional method, whereas the CV of the zeta potential of the dispersion before and after dialysis by the method of the present invention was 0.112 and 0.114, respectively. Met.
  • the particle size is 48.3 ⁇ 3.1 nm, which is 1/2 times smaller than that of the prior art.
  • the PdI of the method of the present invention was also smaller than that of Comparative Example 1.
  • the zeta potential was +21.5 ⁇ 1.8 mV, and the modifying effect by STR-R8 was expected, which was considered to be useful for introduction into cells and administration to animals.
  • the microchannel is useful for producing a dispersion and also as a device for stably modifying the dispersion with a functional element.
  • the dispersion obtained by the method of the present invention was smaller than the dispersion obtained in Comparative Example 1 at each CV value of particle size, PdI and zeta potential. This means that according to the method of the present invention, more uniform particles can be prepared, that is, the accuracy of preparation is high.
  • CoQ 10- MITO-Porter obtained in the microchannel was prepared by removing lipid-modified polyethylene glycol from the lipid composition, the obtained liquid became cloudy. Lipid-modified polyethylene glycol is considered to contribute to improving the solubility of CoQ 10- MITO-Porter in the solution.
  • the amount of preparation was also on the order of ⁇ L in Comparative Example 1 (conventional technique), but the method of the present invention allows unlimited preparation, and a large amount of preparation on the order of L can be expected even at the laboratory level.
  • the method of the present invention can be prepared in a shorter time as the preparation time becomes larger. Therefore, the preparation by the method of the present invention using the microchannel could prepare a dispersion of a lipid membrane structure containing a sparingly soluble compound having a small particle size.
  • the dispersion also had a small PdI.
  • the lipid nanoparticle formation time was significantly reduced. Therefore, it is considered that the production time and cost of the preparation are reduced.
  • a continuous dispersion of lipid membrane structures could be obtained.
  • Example 3 3-1 Evaluation of intracellular uptake (1)
  • fluorescent labeling nitrogenbenzoxadiazole (NBD) -DOPE modified with 0.5 mol% of lipid amount
  • Carriers were prepared, uptake into cervical cancer HeLa cells was evaluated using flow cytometry (FACS), and intracellular localization was observed with a confocal laser scanning microscope (CLMS). Evaluation of cell uptake using flow cytometry showed that the dispersion obtained by the method of the present invention had a large amount of uptake into cells as compared with the conventional method (Fig. 8).
  • Example 4 Change in CoQ 10 concentration
  • the CoQ 10 concentration is halved. It was verified by doubling (0.75 mM, hereinafter, 1/2 CoQ10) and doubling (3 mM, hereinafter, 2 CoQ10).
  • the ethanol dilution concentration in the microchannel was 20%.
  • the particle size before dialysis was the smallest in the concentration of Example 1 (1 CoQ 10 ), 70.5 ⁇ 0.3 nm, and the PdI was also the smallest (FIG. 13).
  • the particle size of all CoQ 10 concentrations was about 50 nm, and the PdI was about 0.2, which tended to increase from that before dialysis.
  • the CoQ 10 concentration was 58.1 ⁇ 5.0 ⁇ M before 1/2 CoQ10 dialysis, 36.0 ⁇ 1.5 ⁇ M after 1/2 CoQ10 dialysis, and 113.3 ⁇ 12.7 ⁇ M before CoQ10 dialysis, respectively. It was 76.1 ⁇ 7.5 ⁇ M after CoQ10 dialysis, 261.8 ⁇ 14.8 ⁇ M before CoQ10 dialysis, and 176.4 ⁇ 10.8 ⁇ M after CoQ10 dialysis.
  • the recovery rates were 83.3 ⁇ 7.1% before 1/2 CoQ10 dialysis, 67.2 ⁇ 3.4% after 1/2 CoQ10 dialysis, and 81.2 ⁇ 9.1% before 1 CoQ10 dialysis, respectively. 1, 72.8 ⁇ 6.7% after CoQ10 dialysis, 93.8 ⁇ 5.3% before 2 CoQ10 dialysis, and 80.5 ⁇ 4.7% after 2 CoQ10 dialysis.
  • the Drag / Lipid (w / w) is 0.10 ⁇ 0.01 before 1/2 CoQ10 dialysis, 0.09 ⁇ 0.00 after 1/2 CoQ10 dialysis, and 0.20 before 1 CoQ10 dialysis, respectively.
  • Example 5 Comparison by Preparation Amount It was verified whether the physical properties changed depending on the preparation amount between the conventional method (Comparative Example 1) and the method of the present invention (preparation method using a microchannel).
  • the particle size, PdI, and zeta potential (ZP) fluctuate greatly depending on the amount prepared at one time.
  • both the particle size and PdI were less affected by the amount of preparation as compared with the conventional method, and stable preparation was possible (Table 2).
  • the unit in order to perform stable modification of STR-R8, the unit must be as small as 100 ⁇ L.
  • the zeta potential becomes 9.8 ⁇ 0.9 mV, which is lower than that at the time of preparation of a small volume. This is a major barrier to scaling up lipid nanoparticle drugs.
  • the zeta potential shows a good value of about 20 mV regardless of the prepared amount, and the CV value (index of variation) is 0.072 before dialysis and 0.072 after dialysis. It is 0.031, which is smaller than the conventional method. From the above, it was shown that the preparation method using the microchannel can stably modify functional elements such as STR-R8 and is indispensable for the production of lipid nanoparticle pharmaceuticals.
  • Example 6 6-1 Concentration of CoQ 10- MITO-Porter An attempt was made to concentrate the dispersion obtained in Example 1. In the conventional method, the CoQ 10 concentration can be concentrated to 506.9 ⁇ 134.1 ⁇ M, but the ultrafiltration filter is likely to be clogged, and an efficient concentration operation may not be possible. Therefore, in this example, in order to increase the drug titer per lipid nanoparticles, an attempt was made to prepare a high-concentration CoQ 10- MITO-Porter by concentrating the dispersion obtained in Example 1. .. The post-dialysis solution of the dispersion obtained in Example 1 was applied to Amicon (MWCO: 100 kDa) and ultrafiltered (1000 g, 25 ° C., 25 minutes).
  • Amicon MWCO: 100 kDa
  • Example 7 Example 7-1. Effect of improving mitochondrial respiration by CoQ 10- MITO-Porter of the present invention
  • MELAS fibroblasts with mitochondrial dysfunction Mitochondrial respiration was evaluated using Leight fibroblasts, LHON fibroblasts, and normal fibroblasts.
  • the number of cells was 2.5 ⁇ 10 4 cells / well for MELAS fibroblasts and 2 ⁇ 10 4 cells / well for other cells. The measurement was carried out by a conventional method.
  • the seeds were sown on Agilent Technologies XFp Cell Culture Minilates (Agilent Technologies, Santa Clara, CA, USA) 24 ⁇ 3 hours before the measurement (about 10000 cells / well).
  • a running medium containing a final concentration of 24.75 mM glucose and 4 mM glutamine was prepared by adding 1.0 M Glucose Solution and 200 mM Glutamine Solution (Agilent) in advance to XF DMEM Medium (Agilent). Cells were cultured using this medium (CO 2 free, 37 ° C.) from 1 hour before the measurement, and measured using Agilent Technologies XFp extracellular flux analysers (Agilent Technologies, Santa Clara, CA, USA).
  • FCCP conjugating agent + 3 mM pyruvate after 15 minutes, measure maximal respiration, and after 15 minutes 0.5 ⁇ M rotenone and 0.5 ⁇ M antimycin.
  • A an inhibitor of electron transport chain respiratory chain complexes I and III was added and baseline oxygen consumption was measured again.
  • the maximum respiratory capacity was determined by subtracting the oxygen consumption rate (%) after the addition of rotenone and antimycin A from the maximum value of the oxygen consumption rate (%).
  • the oxygen consumption rate (%) over time is shown in FIG. 17A, and the maximum respiratory capacity is shown in FIG. 17B.
  • FIGS. 17A and 17B The results were as shown in FIGS. 17A and 17B.
  • PBS of CoQ 10 unencapsulated (-) in the addition of the suspension oxygen consumption rate also improves the maximum breathing capacity is not observed in normal cells.
  • the addition of a PBS (-) suspension of CoQ 10 showed a slight improvement in oxygen consumption and maximal respiration.
  • CoQ 10- MITO-Porter produced using the microchannel greatly improved the oxygen consumption rate and maximum respiration capacity of each cell type.
  • Acetaminophen was dissolved in PBS ( ⁇ ) under heating conditions of 60 ° C.
  • PBS (-) containing acetaminophen (Fuji Film Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered at a dose.
  • CoQ 10- MITO-Porter or PBS (-) (negative control) of the present invention obtained in the microchannel was administered at a dose of 8 ⁇ L / g, respectively, and after 24 hours, cardiac blood sampling and liver removal were performed. It was.
  • the collected blood was left at room temperature for 1 to 2 hours and centrifuged (4 ° C., 15 minutes, 3500 rpm). Then, serum was collected and ALT was measured using transaminase CII-Test Wako (Fuji Film Wako Pure Chemical Industries, Ltd., Osaka, Japan) to evaluate the degree of liver damage.
  • the results were as shown in the left panel of FIG. 18B. As shown in the left panel of FIG. 18B, the amount of ALT, which is a marker of liver damage, was less than 1000 IU / L in the negative control, whereas the CoQ 10- MITO of the present invention obtained by the microchannel was obtained. -The ALT value was significantly reduced in the Porter-administered group.
  • the removed liver was subjected to histological evaluation.
  • the liver was drained with 4% paraformaldehyde (Fuji Film Wako Pure Chemical Industries, Ltd.) and allowed to stand at 4 ° C. overnight. Then, every 4 hours, the liver was treated with a solution containing 10% sucrose, 20% sucrose, and 30% sucrose in order at 4 ° C., and then the liver was allowed to stand overnight with a solution containing 30% sucrose.
  • the liver tissue was placed in an implantation dish, and the tissue section implanter O. C. T. It was filled with Compound (Sakura Finetech Japan Co., Ltd.). In addition, liquid nitrogen was used to freeze the tissue.
  • Frozen samples were sliced with LEICA CM3050S (Leica Biosystems) to a thickness of 20 ⁇ m to prepare frozen sections.
  • the excised tissue sections were thoroughly dried and subjected to hematoxylin-eosin staining (HE staining).
  • HE staining hematoxylin-eosin staining
  • the tissue sections were treated with hematoxylin for 8 minutes, stained, and subjected to running water for 10 minutes.
  • the tissue sections were then treated with eosin for 3 minutes, stained and lightly washed with water.
  • the tissue section was treated with 70% ethanol, 90% ethanol, and 100% ethanol solution in this order, and dehydrated. Further, the tissue section was immersed in xylene.
  • tissue sections were encapsulated using a soft mount. Observation of the tissue section was performed using a stereomicroscope TYPE101M (SHIMADZU). The results were as shown in the right panel of FIG. 18B.
  • a non-hollow lipid structure containing a poorly water-soluble compound could be prepared.
  • CoQ 10 was used as the poorly water-soluble compound
  • CoQ 10 was introduced into the cells and could improve the respiratory ability of the cells.
  • Hepatic disorder could be treated therapeutically in liver disorder model mice.
  • the obtained lipid structure could be imparted with cell membrane permeability, mitochondrial directivity could be imparted, and the introduced CoQ 10 was functionally effective. ..
  • Example 8 In this example, curcumin was used as the poorly water-soluble compound.
  • the prepared particles were dialyzed as described in Example 1 to obtain a solution containing curcumin-containing lipid nanoparticles as curcumin-MITO-Porter.
  • Curcumin is yellow and the residue of curcumin in the solution can be visually confirmed. Since curcumin outside the nanoparticles is removed by solution replacement by dialysis, it is considered that curcumin remains only in the nanoparticles after dialysis.
  • yellow color is confirmed after dialysis, the yellow color reflects curcumin incorporated in the nanoparticles, and the amount of curcumin incorporated in the nanoparticles can be estimated from the shade of yellow.
  • the final dilution concentration of ethanol on the microchannel device was set to 10%, 20%, 30%, and 40%, curcumin-containing lipid nanoparticles were prepared, and the color of the solution was confirmed before and after dialysis. As a result, it was observed that the yellow coloring tended to be very light at the final ethanol dilution concentration of 10%, and the coloring tended to be dark at 20% or more.
  • the particle size and PDI of the curcumin-containing lipid nanoparticles obtained by the DLS method were measured.
  • the results were as shown in FIG. As shown in FIG. 19, after dialysis, curcumin-containing lipid nanoparticles having particularly good PDI were obtained at final ethanol dilutions of 30% and 40%, with a PDI of 0.3 at a final ethanol dilution of 40%. It was smaller. Further, nanoparticles having a particle size of 100 nm to 150 nm were obtained at final ethanol dilution concentrations of 30% and 40%.
  • the lipid membrane structure containing a phospholipid and a lipid-modified uncharged hydrophilic polymer can be freely used not only for CoQ 10 but also for inclusion of a poorly water-soluble compound such as curcumin. Became clear.
  • the lipid membrane structure containing phospholipids and lipid-modified uncharged hydrophilic polymers is non-hollow lipid nanoparticles and has a lipid membrane structure inside the particles. Then, it is considered that the poorly water-soluble compound is incorporated into the lipid membrane structure inside the lipid nanoparticles.
  • the present invention is useful in the field related to the production of lipid nanoparticles of poorly water-soluble compounds.

Abstract

The present invention provides a lipid nanoparticle manufacturing method that allows mass production and can, with good reproducibility, use a MITO-Porter to make a poorly water soluble compound including CoQ10 into lipid nanoparticles having a desired smaller particle size, having a desired polydispersity index, and having a desired zeta potential.

Description

脂質膜構造体及びその製造方法Lipid membrane structure and its manufacturing method
　本発明は、脂質膜構造体及びその製造方法に関する。 The present invention relates to a lipid membrane structure and a method for producing the same.
　近年、ミトコンドリアの機能異常と種々の疾患（神経変性疾患、心筋症、糖尿病、他）との関連が報告されている。組織傷害時の細胞死には、過剰に発生する活性酸素やエネルギー産生低下が大きな要因となっており、これらの機能を制御するミトコンドリアは組織傷害を原因とする種々の疾患（虚血性心疾患、虚血線肝疾患、薬剤性心疾患、薬剤性肝疾患、コエンザイムＱ１０（ＣｏＱ１０）欠乏性腎症、他）に対する有効な治療法になり得る。臓器の虚血状態の回復処置は、様々な疾患治療に適応可能な有用な戦略となる。例えば、外科手術時の一時的な臓器（肝臓、心臓、脳など）の虚血状態は、術中及び術後の患者状態に大きく影響を与える。また、心筋梗塞や脳梗塞などの虚血性心疾患は、平時の予防および発症時の迅速な虚血状態の治療が重要となる。これまでに、虚血による組織傷害の予防及び/又は治療のためのＣｏＱ１０（ノイキノン錠（Ｒ））などが開発されているが、ＣｏＱ１０の高い難水溶性から経口投与では吸収が制限され、治療効果が十分ではない。 In recent years, the association between mitochondrial dysfunction and various diseases (neurodegenerative diseases, cardiomyopathy, diabetes, etc.) has been reported. Excessive active oxygen and decreased energy production are major factors in cell death during tissue damage, and mitochondria that control these functions are various diseases caused by tissue damage (ischemic heart disease, imagination). Chisen liver disease, drug-induced heart disease, drug-induced liver disease, coenzyme Q 10 (CoQ 10) deficiency nephropathy, may be an effective treatment for other). Restoration of ischemic conditions in organs is a useful strategy that can be applied to the treatment of various diseases. For example, the ischemic state of temporary organs (liver, heart, brain, etc.) during surgery has a great influence on the patient's condition during and after surgery. In addition, for ischemic heart disease such as myocardial infarction and cerebral infarction, it is important to prevent it in normal times and to treat the ischemic state promptly at the onset. So far, CoQ 10 (Neuquinone Tablets (R) ) for the prevention and / or treatment of tissue injury due to ischemia has been developed, but absorption is restricted by oral administration due to the high water solubility of CoQ 10. , The therapeutic effect is not enough.
　ＣｏＱ１０は脂溶性が非常に高く注射製剤などに適応できないため、主な剤形は錠剤である。しかし、一般的に錠剤は迅速な対応が求められる虚血性疾患治療への適応は難しく、剤型が錠剤であるが故にＣｏＱ１０には強力な抗酸化作用などの機能があるにも関わらず、適応疾患の幅を狭めている。このような状況に対し、本発明者らはＣｏＱ１０を脂質二重膜小胞（リポソーム）に封入し、溶液中での分散性を獲得させ、さらにミトコンドリア標的化能を付与したナノカプセルとしてＣｏＱ１０搭載ＭＩＴＯ－Ｐｏｒｔｅｒを構築した（非特許文献１、特許文献１）。これらの文献においてＭＩＴＯ－Ｐｏｒｔｅｒとして開示されたＣｏＱ１０搭載製剤は、ジオレイルホスファチジルエタノールアミンとホスファチジン酸又はスフィンゴミエリンとを含有する脂質膜を備え、オクタアルギニン（Ｒ８）を含有する脂質膜構造体からなる。本システムは活性酸素の発生源であるミトコンドリアへの効率的なＣｏＱ１０送達を実現し、虚血時の傷害を抑制することができる。非特許文献２では、肝臓虚血モデル動物（マウス）に静脈投与することにより、当該動物の組織において、ＣｏＱ１０の細胞内への送達およびミトコンドリアへの蓄積を確認している（非特許文献２）。 Since CoQ 10 is very lipophilic and cannot be applied to injection preparations, the main dosage form is tablets. However, in general, it is difficult to adapt tablets to the treatment of ischemic diseases that require prompt response, and despite the fact that CoQ 10 has functions such as strong antioxidant activity because the dosage form is tablets. The range of indications is narrowing. In response to this situation, the present inventors encapsulated CoQ 10 in lipid bilayer vesicles (liposomes) to obtain dispersibility in a solution, and further provided CoQ as a nanocapsule having a mitochondrial targeting ability. A 10- mounted MITO-Porter was constructed (Non-Patent Document 1, Patent Document 1). CoQ 10 mounted formulations disclosed as MITO-Porter in these references, includes a lipid membrane containing a dioleyl phosphatidyl ethanolamine and phosphatidic acid or sphingomyelin, lipid membrane structure containing octaarginine (R8) Become. This system realizes efficient delivery of CoQ 10 to mitochondria, which is a source of active oxygen, and can suppress injury during ischemia. In Non-Patent Document 2, by intravenous administration to the liver ischemia model animals (mice), in the tissue of the animal, it was confirmed delivery and accumulation in the mitochondria into the cell CoQ 10 (Non-Patent Document 2 ).
　リポソームのようなナノカプセルを用いた薬剤送達システム（ＤＤＳ）は、既存薬や核酸医薬品を内封または粒子表面へ担持させることにより、薬物は標的部位に集積し、治療効果の増大や副作用の軽減等の生物学的な効果をもたらす。さらに、ナノカプセルは可溶化技術にも応用され、ＣｏＱ１０のような難水溶性分子の分散性の向上に寄与する。そして封入薬物を酸化から保護する作用もあり、安定性を改善することができ、物理化学的な効果をもたらす。 Drug delivery system (DDS) using nanocapsules such as liposomes allows existing drugs and nucleic acid drugs to be encapsulated or carried on the surface of particles, so that the drugs accumulate at the target site, increasing the therapeutic effect and reducing side effects. It brings about biological effects such as. Furthermore, nanocapsules are also applied to solubilization technology and contribute to improving the dispersibility of poorly water-soluble molecules such as CoQ 10 . It also protects the encapsulated drug from oxidation, can improve stability, and has a physicochemical effect.
　このようにナノカプセル製剤は、従来製剤とは異なる魅力的な新しい物性を実現するため、研究が盛んに行われている。リポソームの代表的な調製法として単純水和法、逆相蒸発法　（ｒｅｖｅｒｓｅ　ｐｈａｓｅ　ｅｖａｐｏｒａｔｉｏｎ　ｖｅｓｉｃｌｅ　（ＲＥＶ）法）およびアルコール希釈法が挙げられる。上記ＣｏＱ１０搭載ＭＩＴＯ－Ｐｏｒｔｅｒをエタノール希釈法で調製した際に、最もＣｏＱ１０の搭載率（脂質に対する薬物の含有量）の高い粒子を得られることができることが示されている（非特許文献３）。 In this way, nanocapsule preparations are being actively researched in order to realize attractive new physical properties different from conventional preparations. Typical methods for preparing liposomes include a simple hydration method, a reverse phase evaporation method (REV) method, and an alcohol dilution method. It has been shown that when the above-mentioned CoQ 10- loaded MITO-Porter is prepared by an ethanol dilution method, particles having the highest CoQ 10 loading rate (drug content with respect to lipid) can be obtained (Non-Patent Document 3). ).
　ＣｏＱ１０以外でも、近年開発候補となる化合物は水溶性が極めて低い化合物が多い。これらの化合物の多くはＢＣＳ　クラス４　に属する。ＢＣＳ　分類（Ｂｉｏｐｈａｒｍａｃｅｕｔｉｃｓ　Ｃｌａｓｓｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ，　Ｔａｂｌｅ　１）とは水溶性と消化管膜透過性の大小の組み合わせに基づく薬物分類である。ＢＣＳ　クラス４　に属する薬物は難水溶解性であることに加えて、難消化管膜透過性であるために消化管吸収性が悪く、一般的に製剤化が難しいと考えられている。医薬品開発では、注射剤開発から始まり、バイオアベイラビリティがよいものを経口剤に剤形変更することが主流である。しかし、ＢＣＳ　クラス４薬物は、難水溶性であるために注射剤化することが困難であり、難消化管膜透過性であるために経口剤として錠剤化して高い効果を発揮させることも困難である。 Other than CoQ 10, developed recently become candidates compound is often very low compound water-soluble. Many of these compounds belong to BCS class 4. The BCS classification (Biopharmaceutics Classication System, Table 1) is a drug classification based on a combination of water solubility and gastrointestinal membrane permeability. In addition to being sparingly soluble in water, drugs belonging to BCS class 4 have poor gastrointestinal absorption due to poor gastrointestinal membrane permeability, and are generally considered to be difficult to formulate. In drug development, it is the mainstream to start with the development of injections and change the dosage form of those with good bioavailability to oral preparations. However, since BCS class 4 drugs are poorly water-soluble, it is difficult to make them into injections, and because they are poorly permeable to the gastrointestinal membrane, it is difficult to tablet them as oral preparations to exert a high effect. is there.
日本特許第５０６７７３３号公報Japanese Patent No. 5067733 ＷＯ２０１８／１９０４２３　Ａ１WO2018 / 190423 A1
　上記のようにｉｎ　ｖｉｖｏの検証から、ＣｏＱ１０搭載ＭＩＴＯ－Ｐｏｒｔｅｒは治療効果が期待できる。しかし、ミトコンドリアへの効率的なＣｏＱ１０送達のためには、より小さくかつ均一な粒子径を有することが望まれる。従来法であるエタノール希釈法（非特許文献２、３）では、同一条件で繰り返し実施しても得られるＣｏＱ１０搭載ＭＩＴＯ－Ｐｏｒｔｅｒの粒子径は、８０～１２０ｎｍの範囲であり、粒子径の均一性の指標である多分散性指数（ＰＤＩ）は０．２～０．４の範囲でばらつき、したがって、調製の再現性が悪く、安定した性能のナノカプセルの供給が困難であった。さらに、ＣｏＱ１０搭載ＭＩＴＯ－Ｐｏｒｔｅｒは、Ｒ８（オクタアルギニン）に代表されるポリアルギニンペプチドで表面修飾され、所定のゼータ電位（例えば、１５～２５ｍＶの範囲）を有するように表面電荷が調整され得る。しかし、エタノール希釈法ではナノカプセル調製後にポリアルギニンペプチドで表面修飾するが、表面電荷の再現性が悪く、かつ所定のゼータ電位を有するナノカプセルを安定的に調製することが難しかった。 From the verification of in vivo as described above, CoQ 10 mounted MITO-Porter is the therapeutic effect can be expected. However, for efficient delivery of CoQ 10 to mitochondria, it is desired to have a smaller and uniform particle size. In the conventional ethanol dilution method (Non-Patent Documents 2 and 3), the particle size of the CoQ 10- equipped MITO-Porter obtained repeatedly under the same conditions is in the range of 80 to 120 nm, and the particle size is uniform. The polydispersity index (PDI), which is an index of sex, varied in the range of 0.2 to 0.4, and therefore the reproducibility of preparation was poor, and it was difficult to supply nanocapsules with stable performance. Further, the CoQ 10- equipped MITO-Porter can be surface-modified with a polyarginine peptide typified by R8 (octaarginine), and the surface charge can be adjusted so as to have a predetermined zeta potential (for example, in the range of 15 to 25 mV). .. However, in the ethanol dilution method, the surface is modified with a polyarginine peptide after the preparation of the nanocapsules, but the reproducibility of the surface charge is poor and it is difficult to stably prepare the nanocapsules having a predetermined zeta potential.
　さらに、エタノール希釈法では、一度の調製で得られるＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの調製量も少なかった　（例えば、約４００　μＬ）。ＣｏＱ１０搭載ＭＩＴＯ－Ｐｏｒｔｅｒを臨床に適応するためには、効率よく封入ができ、安定的に、かつ従来法で得られるよりさらに粒子径が小さいナノカプセルを調製することができる新たな方法の提供が望まれている。 Furthermore, in the ethanol dilution method, the amount of CoQ 10- MITO-Porter prepared in one preparation was also small (for example, about 400 μL). In order to clinically adapt the CoQ 10- equipped MITO-Porter, a new method capable of efficiently encapsulating, stable, and preparing nanocapsules having a smaller particle size than that obtained by the conventional method can be provided. Is desired.
　ＭＩＴＯ－Ｐｏｒｔｅｒは難水溶性化合物を内包することで、効率よく可溶化でき、かつミトコンドリアへの効率的な移送手段となり得る。ＣｏＱ１０以外の難水溶性分子についても、効率よく封入ができ、安定的に、かつ従来法で得られるよりさらに粒子径が小さいナノカプセルを調製することができれば、ＢＣＳクラス４に属する多く難水溶性化合物の医薬への応用が大きく拡がり得る。 By including a poorly water-soluble compound, MITO-Porter can be efficiently solubilized and can be an efficient means of transport to mitochondria. If it is possible to efficiently encapsulate poorly water-soluble molecules other than CoQ 10 and to prepare nanocapsules that are stable and have a smaller particle size than that obtained by the conventional method, many water-resistant molecules belonging to BCS class 4 are poorly water-soluble. The application of sex compounds to pharmaceuticals can be greatly expanded.
　本発明者らは、ＣｏＱ１０を含むＢＣＳクラス４に分類されるような難水溶性化合物を含み、より小さい粒子径、及び/又は、より小さい多分散性指数を有する脂質膜構造体（または脂質ナノ粒子）を再現性良く調製でき、かつ大量生産も可能な脂質膜構造体（または脂質ナノ粒子）の製造方法を提供する。 We have a lipid membrane structure (or lipid) that contains a poorly water-soluble compound such as that classified as BCS Class 4 containing CoQ 10 and has a smaller particle size and / or a smaller polydispersity index. Provided is a method for producing a lipid film structure (or lipid nanoparticles) capable of preparing (nanoparticles) with good reproducibility and mass production.
　本発明は、以下の通りである。
［１］
難水溶性化合物を含有し、動的光散乱（ＤＬＳ）法で測定した平均粒子径が６０ｎｍ以下である脂質膜構造体を分散質として分散媒中に含有する分散体であって、前記脂質膜構造体の脂質膜は、リン脂質及び脂質修飾非電荷親水性ポリマー（好ましくは、脂質修飾ポリエチレングリコール）を含有する、前記分散体。
［２］
前記リン脂質は、ジオレイルホスファチジルエタノールアミンとホスファチジン酸及び／又はスフィンゴミエリンである、［１］に記載の分散体。
［３］
前記難水溶性化合物がＢＣＳ（Ｂｉｏｐｈａｒｍａｃｅｕｔｉｃｓ　Ｃｌａｓｓｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ）　クラス４に属する化合物である、［１］又は［２］に記載の分散体。
［４］
前記難水溶性化合物がＣｏＱ１０である、［１］又は［２］に記載の分散体。
［５］
前記分散媒が水系溶媒である、［１］～［４］のいずれかに記載の分散体。
［６］
前記分散媒はアルコールを含有しない、［１］～［５］のいずれかに記載の分散体。
［７］
前記脂質膜構造体の脂質膜は、膜透過性ペプチドをさらに含有する、［１］～［６］のいずれかに記載の分散体。
［８］
前記膜透過性ペプチドが、連続した４～２０個のアルギニン残基からなるポリアルギニンペプチドである、［７］に記載の分散体。
［９］
ＤＬＳ法で測定した脂質膜構造体の多分散性指数（ＰＤＩ）は、０．３以下である、［１］～［８］のいずれかに記載の分散体。
［１０］
脂質膜構造体のゼータ電位は、１５～２５ｍＶの範囲である、［１］～［９］のいずれかに記載の分散体。
［１１］
前記分散体は、前記難水溶性化合物を細胞のミトコンドリアに移送するために用いられる、［１］～［１０］のいずれかに記載の分散体。
［１２］
リン脂質、膜透過性ペプチド、脂質修飾ポリエチレングリコール並びに難水溶性化合物を溶解したアルコール溶液と水系溶媒とをマイクロ流路構造体のマイクロ流路の入口に連続的に供給し、マイクロ流路内で前記アルコール溶液を水系溶媒で希釈して、難水溶性化合物を含有する脂質膜構造体を分散質として含有する分散体をマイクロ流路の出口から回収する工程を含む、分散体の製造方法。
［１３］
前記リン脂質が、ジオレイルホスファチジルエタノールアミンとホスファチジン酸及び／又はスフィンゴミエリンであり、前記膜透過性ペプチドが、連続した４～２０個のアルギニン残基からなるポリアルギニンペプチドである、［１２］に記載の製造方法。
［１４］
前記脂質膜構造体は、リン脂質、膜透過性ペプチド、及び脂質修飾ポリエチレングリコールを含有する、［１２］又は［１３］に記載の製造方法。
［１５］
マイクロ流路へのアルコール溶液及び水系溶媒の各供給量を、マイクロ流路の出口から回収される分散体のアルコール濃度が４０％以下になる量に制御する、［１２］～［１４］のいずれかに記載の製造方法。
［１６］
マイクロ流路の出口から回収した分散体からアルコールを除去する工程をさらに含む［１２］～［１５］のいずれに記載の製造方法。
［１７］
アルコールを除去した分散体を濃縮する工程をさらに含む、［１２］～［１６］のいずれに記載の製造方法。
［１８］
各工程を０～３０℃の範囲の温度で実施する、［１２］～［１７］のいずれに記載の製造方法。
［１９］
マイクロ流路構造体は、その上流側において、互いに独立した、第１の流動体を導入する第１導入路と、第２の流動体を導入する第２導入路とが、それぞれ一定長を有して合流し、その下流側に向かって１つの希釈流路を形成しており、前記希釈流路は、少なくともその一部において二次元的に屈曲した流路部位を有し、当該屈曲した流路部位は、これより上流の希釈流路の軸線方向ないしその延長方向をＸ方向と、このＸ方向と垂直に交差する希釈流路の幅方向をＹ方向とし、これより上流の希釈流路の流路幅をｙ０とした場合に、Ｙ方向において対向する希釈流路の両側壁面より交互に、流路中心側に向かって、略Ｙ方向（略＋Ｙ方向、略－Ｙ方向）に、１／２ｙ０以上１ｙ０未満の一定高さｈ１，ｈ２．．．を有し、かつＸ方向に一定幅ｘ１，ｘ２．．．を有して突出し、希釈流路の流路幅を規制する構造子が、一定間隔ｄ１，ｄ２．．．をもって少なくとも２つ以上設けられていることで形成されている流路構造体であり、アルコール溶液を第１導入路に導入し、水系溶媒を第２導入路に導入する、［１２］～［１８］のいずれかに記載の製造方法。
［２０］
　上記［１］～［１１］のいずれかに記載の分散体であって、
　ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、脂質修飾ポリエチレングリコールとを含む、脂質膜構造体を含み、
　脂質膜構造体は、ユビキノンまたはそのミトコンドリアの内外膜間の生合成経路における前駆体を含む、分散体。
［２１］
　上記［１］～［１１］のいずれかに記載の分散体であって、
　ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、
　脂質修飾ポリエチレングリコールとを含む、脂質膜構造体を含み、脂質膜構造体は、クルクミンを含む、分散体。
［２２］
　上記［２０］または［２１］に記載の分散体であって、
　脂質膜構造体は、ＤＬＳ法による平均粒径が２０～１５０ｎｍであり、多分散性指数は０．３以下である、分散体。
［２３］
　上記［２０］～［２２］のいずれかに記載の分散体であって、
　脂質膜構造体が、膜透過性ペプチドを表出する、分散体。
［２４］
　上記［２３］に記載の分散体であって、
　膜透過性ペプチドが、オクタアルギニン（Ｒ８）およびＳ２ペプチドからなる群から選択されるペプチドである、分散体。 The present invention is as follows.
[1]
A dispersion containing a poorly water-soluble compound and having a lipid film structure having an average particle size of 60 nm or less measured by a dynamic light scattering (DLS) method as a dispersoid in a dispersion medium, wherein the lipid film is contained. The dispersion in which the lipid membrane of the structure contains a phospholipid and a lipid-modified uncharged hydrophilic polymer (preferably a lipid-modified polyethylene glycol).
[2]
The dispersion according to [1], wherein the phospholipid is diorail phosphatidylethanolamine, phosphatidic acid and / or sphingomyelin.
[3]
The dispersion according to [1] or [2], wherein the poorly water-soluble compound is a compound belonging to BCS (Biopharmaceutics Classification System) class 4.
[4]
The dispersion according to [1] or [2], wherein the poorly water-soluble compound is CoQ 10 .
[5]
The dispersion according to any one of [1] to [4], wherein the dispersion medium is an aqueous solvent.
[6]
The dispersion according to any one of [1] to [5], wherein the dispersion medium does not contain alcohol.
[7]
The dispersion according to any one of [1] to [6], wherein the lipid membrane of the lipid membrane structure further contains a membrane-permeable peptide.
[8]
The dispersion according to [7], wherein the membrane-permeable peptide is a polyarginine peptide consisting of 4 to 20 consecutive arginine residues.
[9]
The dispersion according to any one of [1] to [8], wherein the polydispersity index (PDI) of the lipid membrane structure measured by the DLS method is 0.3 or less.
[10]
The dispersion according to any one of [1] to [9], wherein the zeta potential of the lipid membrane structure is in the range of 15 to 25 mV.
[11]
The dispersion according to any one of [1] to [10], wherein the dispersion is used for transferring the poorly water-soluble compound to the mitochondria of cells.
[12]
An alcohol solution in which a phospholipid, a membrane-permeable peptide, a lipid-modified polyethylene glycol, and a poorly water-soluble compound are dissolved and an aqueous solvent are continuously supplied to the inlet of the microchannel of the microchannel structure in the microchannel. A method for producing a dispersion, which comprises a step of diluting the alcohol solution with an aqueous solvent and recovering a dispersion containing a lipid film structure containing a poorly water-soluble compound as a dispersoid from an outlet of a microchannel.
[13]
In [12], the phospholipid is dioleylphosphatidylethanolamine and phosphatidic acid and / or sphingomyelin, and the membrane-permeable peptide is a polyarginine peptide consisting of 4 to 20 consecutive arginine residues. The manufacturing method described.
[14]
The production method according to [12] or [13], wherein the lipid membrane structure contains a phospholipid, a membrane-permeable peptide, and a lipid-modified polyethylene glycol.
[15]
Any of [12] to [14], wherein the supply amounts of the alcohol solution and the aqueous solvent to the microchannel are controlled so that the alcohol concentration of the dispersion recovered from the outlet of the microchannel is 40% or less. The manufacturing method described in Crab.
[16]
The production method according to any one of [12] to [15], further comprising a step of removing alcohol from the dispersion recovered from the outlet of the microchannel.
[17]
The production method according to any one of [12] to [16], further comprising a step of concentrating the dispersion from which alcohol has been removed.
[18]
The production method according to any one of [12] to [17], wherein each step is carried out at a temperature in the range of 0 to 30 ° C.
[19]
On the upstream side of the microchannel structure, the first introduction path for introducing the first fluid and the second introduction path for introducing the second fluid, which are independent of each other, each have a constant length. Then, they merge to form one dilution flow path toward the downstream side thereof, and the dilution flow path has a two-dimensionally bent flow path portion at least in a part thereof, and the bent flow path is formed. As for the road portion, the axial direction of the dilution flow path upstream from this or the extension direction thereof is the X direction, and the width direction of the dilution flow path perpendicular to the X direction is the Y direction. When the flow path width is y0, 1 / in the substantially Y direction (approximately + Y direction, approximately −Y direction), alternately from both side walls of the dilution channels facing each other in the Y direction, toward the center side of the channel. Constant height h1, h2 of 2y0 or more and less than 1y0. .. .. And has a constant width x1, x2 in the X direction. .. .. The structure that regulates the flow path width of the dilution flow path is formed at regular intervals d1, d2. .. .. It is a flow path structure formed by providing at least two of them, and the alcohol solution is introduced into the first introduction path, and the aqueous solvent is introduced into the second introduction path, [12] to [18]. ] The manufacturing method according to any one of.
[20]
The dispersion according to any one of the above [1] to [11].
Includes a lipid membrane structure comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleyl phosphatidylethanolamine, and lipid-modified polyethylene glycol.
A lipid membrane structure is a dispersion that contains precursors in the biosynthetic pathway between the inner and outer membranes of ubiquinone or its mitochondria.
[21]
The dispersion according to any one of the above [1] to [11].
One or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and dioleylphosphatidylethanolamine.
A dispersion comprising a lipid membrane structure comprising lipid-modified polyethylene glycol, wherein the lipid membrane structure comprises curcumin.
[22]
The dispersion according to the above [20] or [21].
The lipid membrane structure is a dispersion having an average particle size of 20 to 150 nm by the DLS method and a polydispersity index of 0.3 or less.
[23]
The dispersion according to any one of the above [20] to [22].
A dispersion in which the lipid membrane structure represents a membrane-permeable peptide.
[24]
The dispersion according to the above [23].
A dispersion in which the membrane-permeable peptide is a peptide selected from the group consisting of octaarginine (R8) and S2 peptides.
　本発明によれば、以下の発明が提供される。
（１）動的光散乱法によって測定した平均粒子径が６０ｎｍ以下である脂質膜構造体を含む脂質膜構造体の分散体または当該脂質膜構造体を含む組成物であって、脂質膜構造体は、難水溶性化合物を含有し、かつ、脂質膜構造体の脂質膜は、リン脂質を含む、分散体または組成物。
（２）難水溶性化合物が、ＢＣＳ（Ｂｉｏｐｈａｒｍａｃｅｕｔｉｃｓ　Ｃｌａｓｓｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ）　クラス４に属する化合物である、上記（１）に記載の分散体または組成物。
（３）脂質膜構造体が、動的光散乱法によって測定した多分散性指数（ＰＤＩ）が０．３以下である、上記（１）または（２）に記載の分散体または組成物。
（４）脂質膜が、ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンとを含む、上記（１）～（３）のいずれかに記載の分散体または組成物。
（５）脂質膜構造体は、膜透過性ペプチドをさらに含有し、膜透過性ペプチドを表出する、上記（１）～（４）のいずれかに記載の分散体または組成物。
（６）アルコールを含まない、上記（１）～（５）のいずれかに記載の分散体または組成物。
（７）難水溶性化合物が、ＣｏＱ１０である、上記（１）～（６）のいずれかに記載の分散体または組成物。
（８）脂質膜構造体は、非中空の構造体である、上記（１）～（７）のいずれかに記載の分散体または組成物。
（９）脂質修飾された非電荷親水性ポリマー（例えば、ポリエチレングリコール）をさらに含む、上記（１）～（８）のいずれかに記載の分散体または組成物。 According to the present invention, the following inventions are provided.
(1) A dispersion of a lipid film structure containing a lipid film structure having an average particle size of 60 nm or less measured by a dynamic light scattering method, or a composition containing the lipid film structure, which is a lipid film structure. Is a dispersion or composition containing a poorly water-soluble compound and the lipid membrane of the lipid membrane structure containing phospholipids.
(2) The dispersion or composition according to (1) above, wherein the poorly water-soluble compound is a compound belonging to BCS (Biopharmaceutics Classification System) class 4.
(3) The dispersion or composition according to (1) or (2) above, wherein the lipid membrane structure has a polydispersity index (PDI) of 0.3 or less measured by a dynamic light scattering method.
(4) The dispersion according to any one of (1) to (3) above, wherein the lipid membrane contains one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and dioleylphosphatidylethanolamine. Body or composition.
(5) The dispersion or composition according to any one of (1) to (4) above, wherein the lipid membrane structure further contains a membrane-permeable peptide and expresses the membrane-permeable peptide.
(6) The dispersion or composition according to any one of (1) to (5) above, which does not contain alcohol.
(7) The dispersion or composition according to any one of (1) to (6) above, wherein the poorly water-soluble compound is CoQ 10 .
(8) The dispersion or composition according to any one of (1) to (7) above, wherein the lipid membrane structure is a non-hollow structure.
(9) The dispersion or composition according to any one of (1) to (8) above, further comprising a lipid-modified uncharged hydrophilic polymer (for example, polyethylene glycol).
　本発明によれば、小さい粒子径を有し、及び/又は、小さい多分散性指数を有する脂質ナノ粒子を再現性良く調製でき、かつ大量生産も可能な脂質ナノ粒子の製造方法を提供できる。本発明の製造方法では、膜透過性ペプチドも脂質ナノ粒子作成の際に共存させ、膜透過性ペプチドを含有する脂質ナノ粒子を一段階で作成でき、その結果、得られる脂質ナノ粒子の物性が制御される。また、カチオン性ポリマーによる脂質ナノ粒子表面の修飾によって、脂質ナノ粒子のゼータ電位を安定的に制御できた。 According to the present invention, it is possible to provide a method for producing lipid nanoparticles having a small particle size and / or having a small polydispersity index, which can be prepared with good reproducibility and can be mass-produced. In the production method of the present invention, the membrane-permeable peptide can also coexist when producing the lipid nanoparticles, and the lipid nanoparticles containing the membrane-permeable peptide can be produced in one step, and as a result, the physical properties of the obtained lipid nanoparticles can be improved. Be controlled. Moreover, by modifying the surface of the lipid nanoparticles with the cationic polymer, the zeta potential of the lipid nanoparticles could be stably controlled.
　調製量に関しては、従来技術では１バッチあたりμＬオーダーであったが、本発明の方法では材料を供給し続けることにより無制限に調製が可能で、実験室でもＬオーダーの大量調製が見込める。したがって、マイクロ流路を用いることによって、より均質な粒径を有し、及び/または、より小さな粒径を有する、難水溶性化合物（例えば、ＢＣＳクラス４化合物）を含む脂質膜構造体（脂質ナノ粒子）を大量に調製することが可能となる。 Regarding the amount of preparation, in the conventional technique, it was on the order of μL per batch, but in the method of the present invention, it is possible to prepare indefinitely by continuing to supply the material, and a large amount of preparation on the order of L can be expected even in the laboratory. Therefore, by using a microchannel, a lipid membrane structure (lipid) containing a poorly water-soluble compound (eg, a BCS class 4 compound) having a more homogeneous particle size and / or a smaller particle size. Nanoparticles) can be prepared in large quantities.
　これまで、難水溶性分子を含有する粒子としては、６０μｍ以下の平均粒径を有する粒子を得ることはできなかった。本発明によれば、従来では得られなかった、粒子径が６０ｎｍ以下である粒子径を有する難水溶性分子内包脂質ナノ粒子を提供できる。さらに、この脂質ナノ粒子は、多分散性指数がより小さく、その結果、粒子径が小さいことと相まって細胞及びミトコンドリア内への取り込み効率が高まるという効果も奏する。 Until now, it has not been possible to obtain particles having an average particle size of 60 μm or less as particles containing poorly water-soluble molecules. According to the present invention, it is possible to provide poorly water-soluble molecule-encapsulating lipid nanoparticles having a particle size of 60 nm or less, which has not been obtained in the past. Furthermore, the lipid nanoparticles have a smaller polydispersity index, and as a result, have the effect of increasing the efficiency of uptake into cells and mitochondria in combination with the small particle size.
実施例におけるマイクロ流路デバイスを用いる脂質膜構造体（または脂質ナノ粒子若しくはナノカプセル）の調製方法の概略説明図を示す。A schematic explanatory diagram of a method for preparing a lipid membrane structure (or lipid nanoparticles or nanocapsules) using a microchannel device in an example is shown. 実施例１における、脂質相と水相の流速比を調節した場合の実験結果（透析前）を示す。The experimental result (before dialysis) when the flow velocity ratio of the lipid phase and the aqueous phase was adjusted in Example 1 is shown. 実施例１における、脂質相と水相の流速比を調節した場合の実験結果（透析後）を示す。The experimental result (after dialysis) when the flow velocity ratio of the lipid phase and the aqueous phase was adjusted in Example 1 is shown. 実施例１における、透析温度の影響試験の結果を示す。The result of the influence test of the dialysis temperature in Example 1 is shown. 実施例１における、安定性試験の結果を示す。The result of the stability test in Example 1 is shown. 比較例１で従来法で得られた脂質ナノ粒子の物性を示す。Comparative Example 1 shows the physical properties of the lipid nanoparticles obtained by the conventional method. 実施例２における、従来法（比較例１）と実施例１（透析前及び透析後）で得られた脂質膜構造体（または脂質ナノ粒子）の粒子径、ＰｄＩ及びゼータ電位を示す。The particle size, PdI and zeta potential of the lipid membrane structure (or lipid nanoparticles) obtained by the conventional method (Comparative Example 1) and Example 1 (before and after dialysis) in Example 2 are shown. 実施例３における、子宮頸がんＨｅＬａ細胞を用いてフローサイトメトリー（ＦＡＣＳ）による細胞取り込み評価および共焦点レーザー顕微鏡（ＣＬＭＳ）による細胞内局在観察結果を示す。The cell uptake evaluation by flow cytometry (FACS) and the intracellular localization observation result by confocal laser scanning microscope (CLMS) using cervical cancer HeLa cells in Example 3 are shown. 実施例３における、細胞内局在観察　（ＨｅＬａ細胞）画像を示す。The intracellular localization observation (HeLa cell) image in Example 3 is shown. 実施例３における、細胞内局在観察　（モデル疾患細胞）画像を示す。The intracellular localization observation (model disease cell) image in Example 3 is shown. 実施例３における、細胞内局在観察　（Ｈｕｍａｎ　ＣＤＣ細胞）画像を示す。The intracellular localization observation (Human CDC cell) image in Example 3 is shown. 実施例３における、細胞内局在観察　（Ｈｕｍａｎ　ｐｕｌｍｏｎａｒｙ　ａｒｔｅｒｙ　ｓｍｏｏｔｈ　ｍｕｓｃｌｅ細胞）画像を示す。An image of intracellular localization observation (Human pulponary artery smooth muscle cells) in Example 3 is shown. 実施例４における、ＣｏＱ１０濃度の変化の試験の結果を示す。The result of the test of the change of the CoQ 10 concentration in Example 4 is shown. 実施例６におけるＣｏＱ１０含有脂質膜構造体（または脂質ナノ粒子）の濃縮試験Concentration test of CoQ 10- containing lipid membrane structure (or lipid nanoparticles) in Example 6 実施例６におけるＣｏＱ１０含有脂質膜構造体（または脂質ナノ粒子）の安定性試験の結果を示す。The results of the stability test of the CoQ 10- containing lipid membrane structure (or lipid nanoparticles) in Example 6 are shown. 実施例１におけるＣｏＱ１０含有脂質膜構造体（または脂質ナノ粒子）のネガティブ染色による透過電子顕微鏡像を示す。FIG. 5 shows a transmission electron microscope image of the CoQ 10- containing lipid membrane structure (or lipid nanoparticles) in Example 1 by negative staining. 実施例７－１における各種ミトコンドリア病に罹患した患者から得られた細胞株の酸素消費率（ＯＣＲ）に対するＣｏＱ１０含有脂質膜構造体（または脂質ナノ粒子）の効果を示す。The effect of the CoQ 10- containing lipid membrane structure (or lipid nanoparticles) on the oxygen consumption rate (OCR) of the cell line obtained from the patients suffering from various mitochondrial diseases in Example 7-1 is shown. 実施例７－１における各種ミトコンドリア病に罹患した患者から得られた細胞株の酸素消費率（ＯＣＲ）に対するＣｏＱ１０含有脂質膜構造体（または脂質ナノ粒子）の効果を示す。The effect of the CoQ 10- containing lipid membrane structure (or lipid nanoparticles) on the oxygen consumption rate (OCR) of the cell line obtained from the patients suffering from various mitochondrial diseases in Example 7-1 is shown. 実施例７－２における肝障害モデルマウスのＣｏＱ１０含有脂質膜構造体（または脂質ナノ粒子）による治療実験のスキームを示す。Shows the scheme of therapy experiments with CoQ 10 containing lipid membrane structure of the liver injury model mice in Example 7-2 (or lipid nanoparticle). 実施例７－２における肝障害モデルマウスのＣｏＱ１０含有脂質膜構造体（または脂質ナノ粒子）による治療実験の結果を示す。The results of the treatment experiment with the CoQ 10- containing lipid membrane structure (or lipid nanoparticles) of the liver disorder model mouse in Example 7-2 are shown. 実施例８におけるクルクミン含有脂質膜構造体（または脂質ナノ粒子）の透析前後の平均粒径およびＰｄＩを示す。The average particle size and PdI of the curcumin-containing lipid membrane structure (or lipid nanoparticles) before and after dialysis in Example 8 are shown.
＜分散体の構造＞
　分散体は、分散質と分散媒とを含む組成物である。分散質は、難水溶性化合物を含有する脂質膜構造体であり、分散媒は、水系溶媒であり得る。分散体は、好ましくは、着色または非着色の透明な溶液であり得る。
　分散質は、リン脂質と脂質修飾非電荷親水性ポリマー（例えば、脂質修飾ポリエチレングリコール）を含む脂質膜構造体（または脂質ナノ粒子）である。脂質ナノ粒子は、好ましくは、細胞膜への透過性を促進する膜透過性分子（例えば、ペプチド）を表出していてもよい。 <Structure of dispersion>
The dispersion is a composition containing a dispersoid and a dispersion medium. The dispersoid is a lipid membrane structure containing a poorly water-soluble compound, and the dispersion medium can be an aqueous solvent. The dispersion can preferably be a colored or uncolored clear solution.
The dispersoid is a lipid membrane structure (or lipid nanoparticles) containing a phospholipid and a lipid-modified uncharged hydrophilic polymer (eg, lipid-modified polyethylene glycol). The lipid nanoparticles may preferably represent a membrane-permeable molecule (eg, a peptide) that promotes permeability to the cell membrane.
　脂質膜構造体は、リン脂質に加えて、脂質修飾非電荷親水性ポリマーを含む脂質膜構造を有することにより、分散体における分散質の分散性が向上し、分散体（例えば、溶液であるが）の濁りが防止される。リン脂質としては、下記のリン脂質を用いることができ、好ましくは、ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、を含むことができる。脂質膜構造体は、以下に記載される荷電物質及び／又は膜透過性分子をさらに含んでいてもよい。 The lipid membrane structure has a lipid membrane structure containing a lipid-modified uncharged hydrophilic polymer in addition to phospholipids, whereby the dispersibility of the dispersoid in the dispersion is improved, and the dispersion (for example, a solution) ) Is prevented from becoming turbid. As the phospholipid, the following phospholipids can be used, and preferably, one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and dioleylphosphatidylethanolamine can be contained. The lipid membrane structure may further comprise the charged substances and / or membrane permeable molecules described below.
　本発明で用いられ得る非電荷親水性ポリマーは、分子全体として電荷を有さず、親水性であるポリマーであり得る。非電荷親水性ポリマーとしては、本発明の粒子形成を顕著に阻害しない限り特に限定されないが、例えば、ポリオキサゾリンやポリアルキレングリコールが挙げられる。ポリアルキレングリコールとしては、好ましくはポリエチレングリコールを用いることができる。ポリマーは、例えば、数平均分子量において１千～１５万Ｄａ、好ましくは、１千～５千Ｄａ（例えば、約２千Ｄａ）のポリマーを用いることができる。非電荷親水性ポリマーは、脂質と連結して脂質膜構造体に複合体化させることができる。脂質膜構造体から離脱させやすくする観点では、脂質膜から離脱し易い脂質と連結させることができる。そのような脂質としては、ジアシルグリセロール（例えば、１，２－ジミリストイル－ｓｎ－グリセロール）を用いることができる。脂質修飾非電荷親水性ポリマーは、分散体における濁り防止のために添加され得る。 The uncharged hydrophilic polymer that can be used in the present invention can be a polymer that has no charge as a whole molecule and is hydrophilic. The uncharged hydrophilic polymer is not particularly limited as long as it does not significantly inhibit the particle formation of the present invention, and examples thereof include polyoxazolines and polyalkylene glycols. As the polyalkylene glycol, polyethylene glycol can be preferably used. As the polymer, for example, a polymer having a number average molecular weight of 1,000 to 150,000 Da, preferably 1,000 to 5,000 Da (for example, about 2,000 Da) can be used. The uncharged hydrophilic polymer can be linked to the lipid and complexed into a lipid membrane structure. From the viewpoint of facilitating detachment from the lipid membrane structure, it can be linked to a lipid that is easily detached from the lipid membrane. As such a lipid, diacylglycerol (for example, 1,2-dimyristoyl-sn-glycerol) can be used. Lipid-modified uncharged hydrophilic polymers can be added to prevent turbidity in the dispersion.
　脂質膜構造体は、図１に示されるように、脂質を溶解させた脂質相と水相とをバッフルミキサーを内蔵したマイクロ流路デバイス上で混合することによって、ナノ粒子として得られる。製造法の詳細は後述する通りである。脂質相には、有機溶媒、例えば、アルコールを溶媒として用いることができる。アルコールの中では、生体毒性が顕著に生じさせない限り特に限定されないが例えば、ｔ－ブタノール、１－プロパノール、２－プロパノール及び２－ブトキシエタノールを用いることができ、エタノールが好ましく用いられ得る。脂質層にアルコールを用いる場合、難水溶性化合物は当該アルコールに溶解性を示すものとすることができる。溶解させるために加熱が必要な場合には、加熱して難水溶性化合物をアルコールに溶解させてもよい。内包させる難水溶性化合物の種類に応じて、溶解性を高める観点でアルコールを選択してもよい。 As shown in FIG. 1, the lipid membrane structure is obtained as nanoparticles by mixing a lipid phase in which lipids are dissolved and an aqueous phase on a microchannel device containing a baffle mixer. Details of the manufacturing method will be described later. For the lipid phase, an organic solvent, for example, alcohol can be used as the solvent. Among the alcohols, for example, t-butanol, 1-propanol, 2-propanol and 2-butoxyethanol can be used as long as biotoxicity is not significantly caused, and ethanol can be preferably used. When an alcohol is used for the lipid layer, the poorly water-soluble compound can be made soluble in the alcohol. If heating is required for dissolution, the poorly water-soluble compound may be dissolved in alcohol by heating. Alcohol may be selected from the viewpoint of increasing solubility depending on the type of the poorly water-soluble compound to be included.
　脂質膜構造体（または脂質ナノ粒子）は、リン脂質と脂質修飾非電荷親水性ポリマー（例えば、脂質修飾ポリエチレングリコール）を含む。
　分散質は、ＤＬＳ法で測定した粒径において１５０ｎｍ以下、１４０ｎｍ以下、１３０ｎｍ以下、１２０ｎｍ以下、１００ｎｍ以下、９０ｎｍ以下、８０ｎｍ以下、７０ｎｍ以下、６０ｎｍ以下、好ましくは５５ｎｍ以下、または、より好ましくは５０ｎｍ以下の脂質膜構造体（または脂質ナノ粒子）を含み得る。当該脂質膜構造体（または脂質ナノ粒子）は、ＤＬＳ法で測定した粒径において、１０ｎｍ以上、２０ｎｍ以上、３０ｎｍ以上、４０ｎｍ以上、または５０ｎｍ以上であり得る。
　脂質膜構造体（または脂質ナノ粒子）の数平均粒径は、例えば、３０ｎｍ以上、３５ｎｍ以上、４０ｎｍ以上、４５ｎｍ以上、または５０ｎｍ以上であり得、１５０ｎｍ以下、１４０ｎｍ以下、１３０ｎｍ以下、１２０ｎｍ以下、１１０ｎｍ以下、１００ｎｍ以下、９５ｎｍ以下、９０ｎｍ以下、８５ｎｍ以下、８０ｎｍ以下、７５ｎｍ以下、７０ｎｍ以下、６５ｎｍ以下、または６０ｎｍ以下であり得る。数平均粒径は、ある態様では、３０ｎｍ～１５０ｎｍ以下、例えば、５０ｎｍ～１００ｎｍであり得る。粒径は上述の通り、ＤＬＳ法により求められる粒径であり得る。 Lipid membrane structures (or lipid nanoparticles) include phospholipids and lipid-modified uncharged hydrophilic polymers (eg, lipid-modified polyethylene glycol).
The dispersoid has a particle size measured by the DLS method of 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, preferably 55 nm or less, or more preferably 50 nm. It may include the following lipid membrane structures (or lipid nanoparticles): The lipid membrane structure (or lipid nanoparticles) may have a particle size of 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, or 50 nm or more in the particle size measured by the DLS method.
The number average particle size of the lipid membrane structure (or lipid nanoparticles) can be, for example, 30 nm or more, 35 nm or more, 40 nm or more, 45 nm or more, or 50 nm or more, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, It can be 110 nm or less, 100 nm or less, 95 nm or less, 90 nm or less, 85 nm or less, 80 nm or less, 75 nm or less, 70 nm or less, 65 nm or less, or 60 nm or less. In some embodiments, the number average particle size can be 30 nm to 150 nm or less, for example 50 nm to 100 nm. As described above, the particle size may be the particle size obtained by the DLS method.
　脂質膜構造体（または脂質ナノ粒子）は、ＤＬＳ法によって求められる多分散性指数（ＰＤＩ）が、０．５以下、０．４５以下、０．４以下、０．３５以下、０．３以下、０．２５以下、または０．２以下であり得る。ＰＤＩは、１を最大とし、数値が小さくなるほど粒径が均一になる（粒径分布がシャープになる、及び／又は、単分散になる）ことを意味する。本発明のある好ましい態様では、脂質膜構造体（または脂質ナノ粒子）のＰＤＩは、０．３以下である。 The lipid membrane structure (or lipid nanoparticles) has a polydispersity index (PDI) obtained by the DLS method of 0.5 or less, 0.45 or less, 0.4 or less, 0.35 or less, 0.3 or less. , 0.25 or less, or 0.2 or less. PDI means that 1 is the maximum, and the smaller the value, the more uniform the particle size (the particle size distribution becomes sharper and / or monodisperse). In one preferred embodiment of the invention, the PDI of the lipid membrane structure (or lipid nanoparticles) is 0.3 or less.
　本発明のある態様では、脂質膜構造体（または脂質ナノ粒子）は、
　ＤＬＳ法によって測定される粒径が、３０ｎｍ～１５０ｎｍであり、
　ＤＬＳ法によって求められるＰＤＩが、０．３以下である、脂質膜構造体（または脂質ナノ粒子）を含む。ここで上記粒径は、例えば、３０ｎｍ～４０ｎｍ、４０ｎｍ～１５０ｎｍ、４０ｎｍ～１４０ｎｍ、４０ｎｍ～１３０ｎｍ、４０ｎｍ～１２０ｎｍ、４０ｎｍ～１１０ｎｍ、４０ｎｍ～１００ｎｍ、４０ｎｍ～９０ｎｍ、４０ｎｍ～８０ｎｍ、４０ｎｍ～７０ｎｍ、４０ｎｍ～６０ｎｍ、４０ｎｍ～５０ｎｍ、５０ｎｍ～１５０ｎｍ、５０ｎｍ～１４０ｎｍ、５０ｎｍ～１３０ｎｍ、５０ｎｍ～１２０ｎｍ、５０ｎｍ～１１０ｎｍ、５０ｎｍ～１００ｎｍ、５０ｎｍ～９０ｎｍ、５０ｎｍ～８０ｎｍ、５０ｎｍ～７０ｎｍ、５０ｎｍ～６０ｎｍ、６０ｎｍ～１５０ｎｍ、６０ｎｍ～１４０ｎｍ、６０ｎｍ～１３０ｎｍ、６０ｎｍ～１２０ｎｍ、６０ｎｍ～１１０ｎｍ、６０ｎｍ～１００ｎｍ、６０ｎｍ～９０ｎｍ、６０ｎｍ～８０ｎｍ、または６０ｎｍ～７０ｎｍであり得る。 In some aspects of the invention, the lipid membrane structure (or lipid nanoparticles) is
The particle size measured by the DLS method is 30 nm to 150 nm.
It contains a lipid membrane structure (or lipid nanoparticles) having a PDI determined by the DLS method of 0.3 or less. Here, the particle size is, for example, 30 nm to 40 nm, 40 nm to 150 nm, 40 nm to 140 nm, 40 nm to 130 nm, 40 nm to 120 nm, 40 nm to 110 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm. ~ 60nm, 40nm ~ 50nm, 50nm ~ 150nm, 50nm ~ 140nm, 50nm ~ 130nm, 50nm ~ 120nm, 50nm ~ 110nm, 50nm ~ 100nm, 50nm ~ 90nm, 50nm ~ 80nm, 50nm ~ 70nm, 50nm ~ 60nm, 60nm ~ 150nm , 60 nm to 140 nm, 60 nm to 130 nm, 60 nm to 120 nm, 60 nm to 110 nm, 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, or 60 nm to 70 nm.
　本発明のある態様では、脂質膜構造体（または脂質ナノ粒子）は、
　ＤＬＳ法によって測定される粒径が、３０ｎｍ～１５０ｎｍであり、
　ＤＬＳ法によって求められるＰＤＩが、０．３以下である、脂質膜構造体（または脂質ナノ粒子）を５０％以上、６０％以上、７０％以上、または８０％以上含む。ここで上記粒径は、例えば、３０ｎｍ～４０ｎｍ、４０ｎｍ～１５０ｎｍ、４０ｎｍ～１４０ｎｍ、４０ｎｍ～１３０ｎｍ、４０ｎｍ～１２０ｎｍ、４０ｎｍ～１１０ｎｍ、４０ｎｍ～１００ｎｍ、４０ｎｍ～９０ｎｍ、４０ｎｍ～８０ｎｍ、４０ｎｍ～７０ｎｍ、４０ｎｍ～６０ｎｍ、４０ｎｍ～５０ｎｍ、５０ｎｍ～１５０ｎｍ、５０ｎｍ～１４０ｎｍ、５０ｎｍ～１３０ｎｍ、５０ｎｍ～１２０ｎｍ、５０ｎｍ～１１０ｎｍ、５０ｎｍ～１００ｎｍ、５０ｎｍ～９０ｎｍ、５０ｎｍ～８０ｎｍ、５０ｎｍ～７０ｎｍ、５０ｎｍ～６０ｎｍ、６０ｎｍ～１５０ｎｍ、６０ｎｍ～１４０ｎｍ、６０ｎｍ～１３０ｎｍ、６０ｎｍ～１２０ｎｍ、６０ｎｍ～１１０ｎｍ、６０ｎｍ～１００ｎｍ、６０ｎｍ～９０ｎｍ、６０ｎｍ～８０ｎｍ、または６０ｎｍ～７０ｎｍであり得る。 In some aspects of the invention, the lipid membrane structure (or lipid nanoparticles) is
The particle size measured by the DLS method is 30 nm to 150 nm.
It contains 50% or more, 60% or more, 70% or more, or 80% or more of lipid membrane structures (or lipid nanoparticles) having a PDI determined by the DLS method of 0.3 or less. Here, the particle size is, for example, 30 nm to 40 nm, 40 nm to 150 nm, 40 nm to 140 nm, 40 nm to 130 nm, 40 nm to 120 nm, 40 nm to 110 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm. ~ 60nm, 40nm ~ 50nm, 50nm ~ 150nm, 50nm ~ 140nm, 50nm ~ 130nm, 50nm ~ 120nm, 50nm ~ 110nm, 50nm ~ 100nm, 50nm ~ 90nm, 50nm ~ 80nm, 50nm ~ 70nm, 50nm ~ 60nm, 60nm ~ 150nm , 60 nm to 140 nm, 60 nm to 130 nm, 60 nm to 120 nm, 60 nm to 110 nm, 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, or 60 nm to 70 nm.
　本発明のある態様では、脂質膜構造体（または脂質ナノ粒子）は、
　ＤＬＳ法によって測定される粒径の数平均（数平均粒径）が、３０ｎｍ～１５０ｎｍであり、
　ＤＬＳ法によって求められるＰＤＩが、０．３以下である、脂質膜構造体（または脂質ナノ粒子）を含む。ここで、数平均粒径は、ここで粒径は、例えば、３０ｎｍ～４０ｎｍ、４０ｎｍ～１５０ｎｍ、４０ｎｍ～１４０ｎｍ、４０ｎｍ～１３０ｎｍ、４０ｎｍ～１２０ｎｍ、４０ｎｍ～１１０ｎｍ、４０ｎｍ～１００ｎｍ、４０ｎｍ～９０ｎｍ、４０ｎｍ～８０ｎｍ、４０ｎｍ～７０ｎｍ、４０ｎｍ～６０ｎｍ、４０ｎｍ～５０ｎｍ、５０ｎｍ～１５０ｎｍ、５０ｎｍ～１４０ｎｍ、５０ｎｍ～１３０ｎｍ、５０ｎｍ～１２０ｎｍ、５０ｎｍ～１１０ｎｍ、５０ｎｍ～１００ｎｍ、５０ｎｍ～９０ｎｍ、５０ｎｍ～８０ｎｍ、５０ｎｍ～７０ｎｍ、５０ｎｍ～６０ｎｍ、６０ｎｍ～１５０ｎｍ、６０ｎｍ～１４０ｎｍ、６０ｎｍ～１３０ｎｍ、６０ｎｍ～１２０ｎｍ、６０ｎｍ～１１０ｎｍ、６０ｎｍ～１００ｎｍ、６０ｎｍ～９０ｎｍ、６０ｎｍ～８０ｎｍ、または６０ｎｍ～７０ｎｍであり得る。 In some aspects of the invention, the lipid membrane structure (or lipid nanoparticles) is
The number average (number average particle size) of the particle size measured by the DLS method is 30 nm to 150 nm.
It contains a lipid membrane structure (or lipid nanoparticles) having a PDI determined by the DLS method of 0.3 or less. Here, the number average particle size is, for example, 30 nm to 40 nm, 40 nm to 150 nm, 40 nm to 140 nm, 40 nm to 130 nm, 40 nm to 120 nm, 40 nm to 110 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm. -80nm, 40nm-70nm, 40nm-60nm, 40nm-50nm, 50nm-150nm, 50nm-140nm, 50nm-130nm, 50nm-120nm, 50nm-110nm, 50nm-100nm, 50nm-90nm, 50nm-80nm, 50nm-70nm , 50 nm to 60 nm, 60 nm to 150 nm, 60 nm to 140 nm, 60 nm to 130 nm, 60 nm to 120 nm, 60 nm to 110 nm, 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, or 60 nm to 70 nm.
　本発明のある好ましい態様では、脂質膜構造体（または脂質ナノ粒子）は、リン脂質と脂質修飾ポリエチレングリコールとを含む。当該脂質膜構造体（ＣＰＰと呼ばれる）に膜透過性を付与するために、当該脂質膜構造体は、細胞膜透過性ペプチドなどのカチオン性のポリマー（例えば、カチオン性アミノ酸のポリマー、例えば、ポリアルギニンまたはポリリジン）を表出していてもよい。カチオン性のポリマーを脂質膜構造体に表出させるためには、カチオン性のポリマーと脂質（特に限定されないが例えば、ミリストイル基）とのコンジュゲートを作製し、当該コンジュゲートの脂質部分で脂質膜構造体にアンカーさせることによって、カチオン性ポリマーを安定的に脂質膜構造体に表出させることができる。膜透過性ペプチドとしては、ｔａｔペプチド（ヒト免疫不全ウイルスのｔａｔタンパク質のＧｅｎＢａｎｋ登録番号：ＡＡＦ３５３６２．１のアミノ酸配列の４８～６０位に対応するペプチド配列を有する）、オリゴアルギニン（Ｒ９）、オリゴリジン（Ｋ１０）などの塩基性アミノ酸に富むペプチド、ペネトラチンなどの塩基性部分と疎水性部分とを有する両親媒性ペプチド、トランスポーチン、ＴＰ１０などのペプチドが挙げられる。膜透過性ペプチドであって、ミトコンドリア指向性を有するペプチドとしては、オクタアルギニン（Ｒ８）、親油性トリフェニルホスホニウムカチオン（Ｌｉｐｏｐｈｉｌｉｃ　ｔｒｉｐｈｅｎｙｌｐｈｏｓｐｈｏｎｉｕｍ　ｃａｔｉｏｎ；ＴＰＰ）若しくはローダミン（Ｒｈｏｄａｍｉｎｅ）１２３などの脂溶性カチオン物質、ミトコンドリア標的配列（Ｍｉｔｏｃｈｏｎｄｒｉａｌ　Ｔａｒｇｅｔｉｎｇ　Ｓｅｑｕｅｎｃｅ；ＭＴＳ）ペプチド（Ｋｏｎｇ，ＢＷ．　ｅｔ　ａｌ．，Ｂｉｏｃｈｉｍｉｃａ　ｅｔ　Ｂｉｏｐｈｙｓｉｃａ　Ａｃｔａ　２００３，１６２５，ｐｐ．９８－１０８）若しくはＳ２ペプチド（Ｓｚｅｔｏ，Ｈ．Ｈ．　ｅｔ　ａｌ．，Ｐｈａｒｍ．Ｒｅｓ．　２０１１，２８，ｐｐ．２６６９－２６７９）などのポリペプチドが挙げられる。ここでＳ２ペプチドとしては、Ｄｍｔ－Ｄ－Ａｒｇ－ＦＫ－Ｄｍｔ－Ｄ－Ａｒｇ－ＦＫ－ＮＨ２｛ここで、Ｄｍｔは、２，６－ジメチルチロシンであり、Ｄ－ＡｒｇはＤ体のアルギニンであり、Ｆは、Ｌ体のフェニルアラニンであり、Ｋは、Ｌ体のリジンである｝が挙げられる。ある態様では、脂質膜構造体（または脂質ナノ粒子）のゼータ電位が５ｍＶ以上、１０ｍＶ以上、１５ｍＶ以上、１６ｍＶ以上、１７ｍＶ以上、１８ｍＶ以上、１９ｍＶ以上、または２０ｍＶ以上、例えば、約２０ｍＶであり得る。 In one preferred embodiment of the invention, the lipid membrane structure (or lipid nanoparticles) comprises phospholipids and lipid-modified polyethylene glycol. In order to impart membrane permeability to the lipid membrane structure (called CPP), the lipid membrane structure is a cationic polymer such as a cell membrane penetrating peptide (eg, a polymer of cationic amino acids, eg, polyarginine). Alternatively, the polymer may be expressed. In order to express the cationic polymer on the lipid membrane structure, a conjugate of the cationic polymer and a lipid (for example, a myristol group) is prepared, and the lipid membrane is formed on the lipid portion of the conjugate. By anchoring to the structure, the cationic polymer can be stably expressed on the lipid membrane structure. The membrane-permeable peptides include tat peptide (having a peptide sequence corresponding to the 48 to 60th position of the amino acid sequence of AAF35362.1, GenBank registration number of tat protein of human immunodeficiency virus), oligoarginine (R9), oligolysine. Examples thereof include peptides rich in basic amino acids such as (K10), amphipathic peptides having a basic portion and a hydrophobic portion such as penetratin, and peptides such as transportin and TP10. Membrane-permeable peptides having mitochondrial orientation include lipophilic cations such as octaarginine (R8), lipophilic triphenylphosphonium cation (TPP) or rhodamine 123, and mitochondria. Target sequence (Mitochondrial Targeting Sequence; MTS) peptide (Kong, BW. Et al., Biochimica et Biophysica Acta 2003, 1625, pp. 98-108) or S2 peptide (Szeto, H. R. 2011, 28, pp. 2669-2679) and the like. Here, as the S2 peptide, Dmt-D-Arg-FK-Dmt-D-Arg-FK-NH 2 {Here, Dmt is 2,6-dimethyltyrosine, and D-Arg is D-form arginine. Yes, F is L-form phenylalanine and K is L-form lysine}. In some embodiments, the zeta potential of the lipid membrane structure (or lipid nanoparticles) can be 5 mV or higher, 10 mV or higher, 15 mV or higher, 16 mV or higher, 17 mV or higher, 18 mV or higher, 19 mV or higher, or 20 mV or higher, for example, about 20 mV. ..
　ある好ましい態様では、本発明の分散体は、分散媒中に、製造途上で用いられ得るアルコールを１％未満、０．５％未満、０．１％未満、または０．０５％未満しか含まないか、またはアルコールを含まない。製造途上で用いられ得るアルコールを含まない分散体を得るためには、例えば、分散体をアルコールを含有しない透析外液で透析することができる。透析外液は、例えば、生理食塩水などの水溶液であり得る。 In certain preferred embodiments, the dispersions of the invention contain less than 1%, less than 0.5%, less than 0.1%, or less than 0.05% alcohol that can be used during production in the dispersion medium. Or does not contain alcohol. In order to obtain an alcohol-free dispersion that can be used in the process of production, for example, the dispersion can be dialyzed with an alcohol-free external dialysis solution. The external dialysis solution can be, for example, an aqueous solution such as physiological saline.
　ある好ましい態様では、本発明の分散体は、難水溶性化合物（例えば、ＢＣＳクラス４の化合物、例えば、クルクミン及び／又はＣｏＱ１０）を内包した脂質膜構造体を含む。脂質膜構造体の作製途上において、エタノールを用いる場合には、難水溶性化合物は、エタノールに溶解性を示す化合物であり得る。例えば、ある好ましい態様において、本発明の分散体では、脂質膜構造体は、脂質膜の総量に対して、ＣｏＱ１０を１０～４０モル％（好ましくは、２０～４０モル％）、例えば、２０～３０モル％）の範囲で含む。 In one preferred embodiment, the dispersion of the present invention comprises a lipid membrane structure containing a poorly water soluble compound (eg, a BCS class 4 compound such as curcumin and / or CoQ 10 ). When ethanol is used in the process of preparing the lipid membrane structure, the poorly water-soluble compound can be a compound exhibiting solubility in ethanol. For example, in a preferred embodiment, in the dispersion of the present invention, the lipid membrane structure contains 10-40 mol% (preferably 20-40 mol%) of CoQ 10 relative to the total amount of lipid membrane, eg 20. Included in the range of ~ 30 mol%).
　ＣｏＱ１０を内包した脂質膜構造体は、ＣｏＱ１０の代わりにユビキノン（酸化型ユビキノン）を用いてもよく、あるいは、ミトコンドリアの内外膜間の生合成経路におけるユビキノンやＣｏＱ１０の前駆体を用いてもよい。ユビキノンは、そのイソプレンの繰り返し単位の個数ｎに対応させて、ＣｏＱｎと呼ばれる。ｎは、本発明では、ｎは４～１５の範囲または６～１２の範囲のいずれかの自然数であり得、例えば、６～１０の範囲の自然数であり得、例えば、８～１２の範囲の自然数であり得、例えば、８～１０の範囲の自然数であり得、例えば、１０であり得る。これらの前駆体としては、例えば、ジメトキシユビキノン（ＤＭＱ）、および５－ヒドロキシユビキノン（５－ＨＱ）が挙げられる。本発明のＲ８表出型脂質膜構造体は、ユビキノンをミトコンドリア内膜へ送達したと考えられ、細胞の酸素消費速度を向上させることができた。したがって、本発明のＲ８表出型脂質膜構造体は、細胞外からミトコンドリア内膜にユビキノンを送達することに用いられ得る。 The lipid membrane structure containing CoQ 10 may use ubiquinone (oxidized ubiquinone) instead of CoQ 10 , or may use a precursor of ubiquinone or CoQ 10 in the biosynthetic pathway between the inner and outer membranes of mitochondria. May be good. Ubiquinone is called CoQ n , corresponding to the number n of repeating units of the isoprene. In the present invention, n can be a natural number in the range of 4 to 15 or 6 to 12, for example, a natural number in the range of 6 to 10, for example, in the range of 8 to 12. It can be a natural number, eg, a natural number in the range 8-10, eg, 10. Examples of these precursors include dimethoxyubiquinone (DMQ) and 5-hydroxyubiquinone (5-HQ). The R8-expressing lipid membrane structure of the present invention was considered to have delivered ubiquinone to the inner mitochondrial membrane, and was able to improve the oxygen consumption rate of cells. Therefore, the R8 expressive lipid membrane structure of the present invention can be used to deliver ubiquinone from extracellular to the inner mitochondrial membrane.
　本発明のある好ましい態様では、分散体（または組成物）は、ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、ステアリル化ポリエチレングリコールとを含む、脂質膜構造体を含み、脂質膜構造体は、ユビキノンまたはそのミトコンドリアの内外膜間の生合成経路における前駆体（好ましくは、ＣｏＱｎ、特にＣｏＱ１０）を含む。脂質膜構造体は、ＤＬＳ法による粒径が２０～１００ｎｍである脂質膜構造体を含み、ＰＤＩは０．３未満である。この好ましい態様における分散体（組成物）は、数平均粒径が５０ｎｍ～７０ｎｍであり得る。この好ましい態様において、分散媒は、生理食塩水であり得、好ましくは、アルコールを１％未満、０．５％以下、０．４％以下、０．３％以下、０．２％以下、０．１％以下、若しくは０．０１％以下、または検出限界以下しか含有しないか、アルコールを含有しない。この態様において脂質膜構造体は、細胞膜透過性ペプチド（例えば、Ｒ８）をさらに表出させていてもよい。脂質膜構造体は、非中空である。 In one preferred embodiment of the invention, the dispersion (or composition) comprises one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleylphosphatidylethanolamine, and steallylated polyethylene glycol. , The lipid membrane structure comprises precursors (preferably CoQ n , particularly CoQ 10 ) in the biosynthetic pathway between the inner and outer membranes of ubiquinone or its mitochondria. The lipid membrane structure includes a lipid membrane structure having a particle size of 20 to 100 nm according to the DLS method, and has a PDI of less than 0.3. The dispersion (composition) in this preferred embodiment can have a number average particle size of 50 nm to 70 nm. In this preferred embodiment, the dispersion medium can be saline, preferably less than 1%, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0 alcohol. .Contains less than 1%, or less than 0.01%, or less than the detection limit, or does not contain alcohol. In this embodiment, the lipid membrane structure may further express a cell membrane penetrating peptide (eg, R8). The lipid membrane structure is non-hollow.
　本発明のある好ましい態様では、分散体（または組成物）は、ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、ステアリル化ポリエチレングリコールとを含む、脂質膜構造体を含み、脂質膜構造体は、クルクミンを含み、脂質膜構造体は、ＤＬＳ法による粒径が４０～３００ｎｍである脂質膜構造体を含み、ＰＤＩは０．３未満である。この好ましい態様における分散体（組成物）は、数平均粒径が１００ｎｍ～１２０ｎｍであり得る。この好ましい態様において、分散媒は、生理食塩水であり得、好ましくは、アルコールを１％未満、０．５％以下、０．４％以下、０．３％以下、０．２％以下、０．１％以下、若しくは０．０１％以下、または検出限界以下しか含有しないか、アルコールを含有しない。この態様において脂質膜構造体は、上記細胞膜透過性ペプチド及び／またはミトコンドリア指向性ペプチド（例えば、Ｒ８若しくはＳ２ペプチドまたはその両方）をさらに表出させていてもよい。 In one preferred embodiment of the invention, the dispersion (or composition) comprises one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleylphosphatidylethanolamine, and steallylated polyethylene glycol. , The lipid membrane structure comprises curcumin, the lipid membrane structure comprises a lipid membrane structure having a particle size of 40-300 nm according to the DLS method, and the PDI is less than 0.3. .. The dispersion (composition) in this preferred embodiment can have a number average particle size of 100 nm to 120 nm. In this preferred embodiment, the dispersion medium can be saline, preferably less than 1%, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0 alcohol. .Contains less than 1%, or less than 0.01%, or less than the detection limit, or does not contain alcohol. In this embodiment, the lipid membrane structure may further express the cell membrane penetrating peptide and / or mitochondrial directional peptide (eg, R8 and / or S2 peptide).
＜分散体の製造方法＞
　本発明の分散体の製造方法は、上記の分散体を製造することに用いられ得る。本発明の分散体の製造方法は、リン脂質、膜透過性ペプチド、脂質修飾ポリエチレングリコール並びに難水溶性化合物を溶解したアルコール溶液と水系溶媒とをマイクロ流路構造体のマイクロ流路の入口に連続的に供給し、マイクロ流路内で前記アルコール溶液を水系溶媒で混合（すなわち、希釈）して、難水溶性化合物を含有する脂質膜構造体を分散質として含有する分散体をマイクロ流路の出口から回収する工程を含む。このようにして得られる脂質膜構造体（分散質）は、脂質ナノ粒子である。また、当該ナノ粒子は、内部がリン脂質または脂質膜で充填された構造体（非中空の構造体；若しくは非中空の脂質ナノ粒子；または、ミトコンドリア指向性分子を表出するものの場合は、非中空のＭＩＴＯ－Ｐｏｒｔｅｒ）であり得る。以下、本発明における分散質を脂質ナノ粒子（またはナノカプセル）と呼ぶこともある。 <Manufacturing method of dispersion>
The method for producing a dispersion of the present invention can be used to produce the above-mentioned dispersion. In the method for producing a dispersion of the present invention, an alcohol solution in which a phospholipid, a membrane-permeable peptide, a lipid-modified polyethylene glycol, and a poorly water-soluble compound are dissolved and an aqueous solvent are continuously connected to the entrance of the microchannel of the microchannel structure. The alcohol solution is mixed (that is, diluted) with an aqueous solvent in the microchannel, and a dispersion containing a lipid membrane structure containing a poorly water-soluble compound as a dispersoid is provided in the microchannel. Includes the step of collecting from the outlet. The lipid membrane structure (dispersant) thus obtained is lipid nanoparticles. In addition, the nanoparticles are non-hollow structures filled with phospholipids or lipid membranes (non-hollow structures; or non-hollow lipid nanoparticles; or non-hollow if they represent mitochondrial directional molecules). It can be a hollow MITO-Porter). Hereinafter, the dispersoid in the present invention may be referred to as lipid nanoparticles (or nanocapsules).
　この方法によれば、難水溶性化合物を含有し、かつＤＬＳ法で測定した平均粒子径が６０ｎｍ以下である脂質膜構造体を含む分散体を得ることもできる。 According to this method, it is also possible to obtain a dispersion containing a poorly water-soluble compound and containing a lipid membrane structure having an average particle size of 60 nm or less measured by the DLS method.
　リン脂質は、特に限定されないが例えば、ホスファチジルコリン（例えば、ジオレオイルホスファチジルコリン、ジラウロイルホスファチジルコリン、ジミリストイルホスファチジルコリン、ジパルミトイルホスファチジルコリン、ジステアロイルホスファチジルコリン等）、ホスファチジルグリセロール（例えば、ジオレオイルホスファチジルグリセロール、ジラウロイルホスファチジルグリセロール、ジミリストイルホスファチジルグリセロール、ジパルミトイルホスファチジルグリセロール、ジステアロイルホスファチジグリセロール等）、ホスファチジルエタノールアミン（例えば、ジオレイルホスファチジルエタノールアミン、ジラウロイルホスファチジルエタノールアミン、ジミリストイルホスファチジルエタノールアミン、ジパルミトイルホスファチジルエタノールアミン、ジステアロイルホスファチジエタノールアミン等）、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジン酸、カルジオリピン、スフィンゴミエリン、セラミドホスホリルエタノールアミン、セラミドホスホリルグリセロール、セラミドホスホリルグリセロールホスファート、１、２－ジミリストイル－１、２－デオキシホスファチジルコリン、プラスマロゲン、卵黄レシチン、大豆レシチン、これらの水素添加物等であることができる。リン脂質は、ホスファチジルエタノールアミン（例えば、ジオレイルホスファチジルエタノールアミン、ジラウロイルホスファチジルエタノールアミン、ジミリストイルホスファチジルエタノールアミン、ジパルミトイルホスファチジルエタノールアミン、ジステアロイルホスファチジエタノールアミン等）、スフィンゴミエリンであることが好ましく、より好ましくはジオレイルホスファチジルエタノールアミン、スフィンゴミエリンである。 The phospholipid is not particularly limited, and is, for example, phosphatidylcholine (for example, dioleoil phosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.) Phosphatidylglycerol, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc. Amin, distearoylphosphatidylethanolamine, etc.), phosphatidylserine, phosphatidylinositol, phosphatidylic acid, cardiolipin, sphingomyelin, ceramide phosphorylethanolamine, ceramidephosphorylglycerol, ceramidephosphorylglycerol phosphate, 1,2-dipalmitoyl-1,2- It can be deoxyphosphatidylcholine, plasmalogen, egg yolk lecithin, soybean lecithin, hydrogenated additives thereof and the like. Phospholipids are preferably phosphatidylethanolamine (eg, diolaylphosphatidylethanolamine, dilauroylphosphatidylethanolamine, dimyristylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, etc.) and sphingomyelin. More preferably, diorail phosphatidylethanolamine and sphingomyelin.
　リン脂質を含む脂質膜は、リン脂質に加えて荷電物質を含有することができ、荷電物質は、脂質膜に正荷電又は負荷電を付与することができる、脂質膜の構成成分であり、脂質膜に含有される荷電物質量は、脂質膜を構成する総物質量の通常３０％（モル比）以下、好ましくは２５％（モル比）以下、さらに好ましくは２０％（モル比）以下である。なお、荷電物質の含有量の下限値は０である。正荷電を付与する荷電物質としては、例えば、ステアリルアミン、オレイルアミン等の飽和又は不飽和脂肪族アミン；ジオレオイルトリメチルアンモニウムプロパン等の飽和又は不飽和カチオン性合成脂質等が挙げられ、負電荷を付与する荷電物質としては、例えば、ジセチルホスフェート、コレステリルヘミスクシネート、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジン酸等が挙げられる。ある態様では、脂質膜構造体（または脂質ナノ粒子）のゼータ電位が５ｍＶ以上、１０ｍＶ以上、１５ｍＶ以上、１６ｍＶ以上、１７ｍＶ以上、１８ｍＶ以上、１９ｍＶ以上、または２０ｍＶ以上、例えば、約２０ｍＶであり得る。 A lipid membrane containing a phospholipid can contain a charged substance in addition to the phospholipid, and the charged substance is a component of the lipid membrane capable of imparting a positive charge or a negative charge to the lipid membrane, and is a lipid. The amount of charged substance contained in the membrane is usually 30% (molar ratio) or less, preferably 25% (molar ratio) or less, and more preferably 20% (molar ratio) or less of the total amount of substances constituting the lipid membrane. .. The lower limit of the content of the charged substance is 0. Examples of the charged substance that imparts a positive charge include saturated or unsaturated aliphatic amines such as stearylamine and oleylamine; saturated or unsaturated cationic synthetic lipids such as dioleoyltrimethylammonium propane, and the like, and negative charges are given. Examples of the charged substance to be imparted include disetyl phosphate, cholesteryl hemisuccinate, phosphatidylserine, phosphatidyl inositol, phosphatidyl acid and the like. In some embodiments, the zeta potential of the lipid membrane structure (or lipid nanoparticles) can be 5 mV or higher, 10 mV or higher, 15 mV or higher, 16 mV or higher, 17 mV or higher, 18 mV or higher, 19 mV or higher, or 20 mV or higher, for example, about 20 mV. ..
　リン脂質は、ジオレイルホスファチジルエタノールアミンとホスファチジン酸及び／又はスフィンゴミエリンであること（すなわち、ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、を含むこと）が、ミトコンドリア内への目的物質である難水溶性化合物の効果的な送達のために好ましい。 The phospholipids are diorail phosphatidylethanolamine and phosphatidic acid and / or sphingomyelin (ie, one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, and diorail phosphatidylethanolamine. Included) is preferred for effective delivery of the poorly water-soluble compound, which is the substance of interest, into the mitochondria.
　膜透過性分子は、例えば、カチオン性のポリマーであり得る。膜透過性分子は、例えば、膜透過性ペプチドであり得る。膜透過性ペプチドは、ミトコンドリア内への目的物質である難水溶性化合物の効果的な送達に有効な膜透過性ドメインである。膜透過性ペプチドは、特許文献１に記載の段落００５２～００９２に記載の膜透過性ペプチドであることができ、好ましくは連続した４～２０個のアルギニン残基からなるポリアルギニンペプチドである。ポリアルギニンペプチドは、好ましくは６～１２個、さらに好ましくは７～１０個の連続したアルギニン残基、または８個の連続したアルギニン残基からなる。膜透過性ペプチドは、脂質と連結させることができる。これにより、脂質膜構造体に膜透過性ペプチドを含有させ、かつ、膜透過性ペプチドを脂質膜構造体上に表出させることができる。ある態様では、脂質膜構造体（または脂質ナノ粒子）のゼータ電位が５ｍＶ以上、１０ｍＶ以上、１５ｍＶ以上、１６ｍＶ以上、１７ｍＶ以上、１８ｍＶ以上、１９ｍＶ以上、または２０ｍＶ以上、例えば、約２０ｍＶであり得る。 The membrane permeable molecule can be, for example, a cationic polymer. The membrane permeable molecule can be, for example, a membrane permeable peptide. Membrane-permeable peptides are membrane-permeable domains that are effective for the effective delivery of poorly water-soluble compounds of interest into mitochondria. The membrane-permeable peptide can be the membrane-permeable peptide described in paragraphs 0052 to 0092 of Patent Document 1, preferably a polyarginine peptide consisting of 4 to 20 consecutive arginine residues. The polyarginine peptide preferably consists of 6-12, more preferably 7-10 contiguous arginine residues, or 8 contiguous arginine residues. Membrane-permeable peptides can be linked to lipids. Thereby, the membrane-permeable peptide can be contained in the lipid membrane structure, and the membrane-permeable peptide can be expressed on the lipid membrane structure. In some embodiments, the zeta potential of the lipid membrane structure (or lipid nanoparticles) can be 5 mV or higher, 10 mV or higher, 15 mV or higher, 16 mV or higher, 17 mV or higher, 18 mV or higher, 19 mV or higher, or 20 mV or higher, for example, about 20 mV. ..
　脂質修飾ポリエチレングリコール（ＰＥＧ）は、脂質膜に親水性を付与する成分である。脂質修飾ポリエチレングリコール（ＰＥＧ）は、ポリエチレングリコール（ＰＥＧ）に脂質修飾した化合物であり、ポリエチレングリコールの分子量は、例えば３００～１０、０００程度、好ましくは５００～１０、０００程度、さらに好ましくは１、０００～５、０００程度である。ＰＥＧの分子量は、数平均分子量で表される。脂質修飾ポリエチレングリコールとしては、例えば、ジオレオイルグリセロール修飾ＰＥＧ、ジラウロイルグリセロール修飾ＰＥＧ、ジミリストイルグリセロール修飾ＰＥＧ、ジパルミトイルグリセロール修飾ＰＥＧ、ジステアロイルグリセロール修飾ＰＥＧ等であることができる。脂質修飾ポリエチレングリコール（脂質修飾ＰＥＧ）は、より具体的には、例えば、ステアリル化ポリエチレングリコール（例えばステアリン酸ＰＥＧ４５（ＳＴＲ－ＰＥＧ４５）など）を用いることができる。その他、Ｎ－［カルボニル－メトキシポリエチレングリコール－２０００］－１、２－ジパルミトイル－ｓｎ－グリセロ－３－ホスフォエタノールアミン、ｎ－［カルボニル－メトキシポリエチレングリコール－５０００］－１、２－ジパルミトイル－ｓｎ－グリセロ－３－ホスフォエタノールアミン、Ｎ－［カルボニル－メトキシポリエチレングリコール－７５０］－１、２－ジステアロイル－ｓｎ－グリセロ－３－ホスフォエタノールアミン、Ｎ－［カルボニル－メトキシポリエチレングリコール－２０００］－１、２－ジステアロイル－ｓｎ－グリセロ－３－ホスフォエタノールアミン、Ｎ－［カルボニル－メトキシポリエチレングリコール－５０００］－１、２－ジステアロイル－ｓｎ－グリセロ－３－ホスフォエタノールアミンなどのポリエチレングリコール誘導体などを用いることもできる。但し、これらに限定されることはない。 Lipid-modified polyethylene glycol (PEG) is a component that imparts hydrophilicity to lipid membranes. Lipid-modified polyethylene glycol (PEG) is a compound obtained by lipid-modifying polyethylene glycol (PEG), and the molecular weight of polyethylene glycol is, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably 1. It is about 000 to 5,000. The molecular weight of PEG is represented by a number average molecular weight. Examples of the lipid-modified polyethylene glycol can be dioleoylglycerol-modified PEG, dilauroylglycerol-modified PEG, dimyristoylglycerol-modified PEG, dipalmitoylglycerol-modified PEG, distearoylglycerol-modified PEG and the like. More specifically, as the lipid-modified polyethylene glycol (lipid-modified PEG), stearyllated polyethylene glycol (for example, PEG45 stearate (STR-PEG45)) can be used. In addition, N- [carbonyl-methoxypolyethylene glycol-2000] -1,2-dipalmitoyl-sn-glycero-3-phoethanolamine, n- [carbonyl-methoxypolyethylene glycol-5000] -1,2-dipalmitoyl -Sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol-750] -1,2-distearoyl-sn-glycero-3-phoethanolamine, N- [carbonyl-methoxypolyethylene glycol -2000] -1,2-Distearoyl-sn-glycero-3-phoethanolamine, N- [carbonyl-methoxypolyethylene glycol-5000] -1,2-distearoyl-sn-glycero-3-phoethanolamine A polyethylene glycol derivative such as amine can also be used. However, it is not limited to these.
　難水溶性化合物は、制限はないが、例えば、Ｂｉｏｐｈａｒｍａｃｅｕｔｉｃｓ　Ｃｌａｓｓｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ（ＢＣＳ）クラス４に属する化合物であることができる。難水溶性化合物は、特に限定されないが、例えば、テルフェナジン（Ｔｅｒｆｅｎａｄｉｎｅ）、フロセミド（Ｆｕｒｏｓｅｍｉｄｅ）、シクロスポリン（Ｃｙｃｌｏｓｐｏｒｉｎ）、アセタゾラミド（Ａｃｅｔａｚｏｌａｍｉｄｅ）、コリスチン（Ｃｏｌｉｓｔｉｎ）、メベンダゾール（Ｍｅｂｅｎｄａｚｏｌｅ）、コエンザイムＱ１０（ＣｏＱ１０）等を挙げることができる。 The poorly water-soluble compound can be, for example, a compound belonging to Biopharmaceutics Classification System (BCS) class 4 without limitation. Poorly water-soluble compound is not particularly limited, for example, terfenadine (Terfenadine), furosemide (furosemide), cyclosporine (Cyclosporin), acetazolamide (acetazolamide), colistin (Colistin), mebendazole (Mebendazole), coenzyme Q 10 (CoQ 10) And so on.
　アルコール溶液のアルコールに特に制限はないが、例えば、エタノール、ｔ－ブタノール、１－プロパノール、２－プロパノール及び２－ブトキシエタノール等を挙げることができる。 The alcohol in the alcohol solution is not particularly limited, and examples thereof include ethanol, t-butanol, 1-propanol, 2-propanol, and 2-butoxyethanol.
　アルコール溶液の各成分の濃度は、所望の脂質膜構造体（または脂質ナノ粒子）に応じて適宜決定することができ、特に限定されないが、
　リン脂質は、例えば、５０～８０ｍｏｌ％の範囲、
　膜透過性ペプチドは、例えば、５～２０ｍｏｌ％の範囲、
　脂質修飾ポリエチレングリコールは、例えば、１～１０ｍｏｌ％の範囲、
　難水溶性化合物は、例えば、１０～４０ｍｏｌ％の範囲
にすることができる。 The concentration of each component of the alcohol solution can be appropriately determined according to the desired lipid membrane structure (or lipid nanoparticles), and is not particularly limited.
Phospholipids are, for example, in the range of 50-80 mol%,
Membrane-permeable peptides are, for example, in the range of 5-20 mol%,
Lipid-modified polyethylene glycols are, for example, in the range of 1-10 mol%.
The poorly water-soluble compound can be in the range of 10 to 40 mol%, for example.
　水系溶媒としては、水溶液が挙げられ、例えば、水、または基本的に水を主成分とする、例えば、生理食塩水、緩衝水溶液（例えば、リン酸緩衝液、酢酸緩衝液、クエン酸緩衝液等）等を挙げることができ、本発明において好ましく用いることができる。本発明のある態様では、リン酸緩衝液が好ましく用いられ得る。 Examples of the aqueous solvent include an aqueous solution, for example, water or basically water as a main component, for example, a physiological saline solution, a buffer aqueous solution (for example, a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, etc.). ), Etc., which can be preferably used in the present invention. In certain aspects of the invention, phosphate buffer may be preferably used.
　本発明の分散体の製造方法においては、リン脂質等を含有するアルコール溶液を水系溶媒で希釈して、難水溶性化合物を含有する脂質膜構造体を分散質として含有する分散体をマイクロ流路の出口から回収する。本発明の分散体の製造方法において用いるマイクロ流路構造体は、リン脂質等を含有するアルコール溶液を流通下、水系溶媒で希釈して、脂質膜構造体を分散質として含有する分散体を調製できるマイクロ流路構造体であれば、特に制限なく利用できる。そのようなマイクロ流路構造体は、例えば、特許５８２３４０５号公報、特許６２３４９７１号公報、非特許文献４、非特許文献５、ＷＯ２０１８／１９０４２３　Ａ１（特許文献２）等に記載のマイクロ流路構造体を例示できる。本発明においては、特に、特許文献２に記載のマイクロ流路構造体を用いることが、脂質膜構造体の粒子径を所望の値に制御しつつ、かつ分散度の小さい粒の揃った脂質膜構造体を分散質として含有する分散体を得ることに有利に用いられ得る。 In the method for producing a dispersion of the present invention, an alcohol solution containing a phospholipid or the like is diluted with an aqueous solvent, and a dispersion containing a lipid membrane structure containing a poorly water-soluble compound as a dispersoid is provided as a microchannel. Collect from the exit of. The microchannel structure used in the method for producing a dispersion of the present invention is prepared by diluting an alcohol solution containing a phospholipid or the like with an aqueous solvent to prepare a dispersion containing a lipid membrane structure as a dispersoid. Any microchannel structure that can be used can be used without particular limitation. Such a microchannel structure is described in, for example, Patent No. 5823405, Japanese Patent No. 6234971, Non-Patent Document 4, Non-Patent Document 5, WO2018 / 190423 A1 (Patent Document 2) and the like. Can be exemplified. In the present invention, in particular, by using the microchannel structure described in Patent Document 2, a lipid membrane having a uniform particle size with a small degree of dispersion while controlling the particle size of the lipid membrane structure to a desired value. It can be advantageously used to obtain a dispersion containing the structure as a dispersoid.
　特許文献２に記載のマイクロ流路構造体は、その上流側において、互いに独立した、第１の流動体を導入する第１導入路と、第２の流動体を導入する第２導入路とが、それぞれ一定長を有して合流し、その下流側に向かって１つの希釈流路を形成しており、前記希釈流路は、少なくともその一部において屈曲（例えば、二次元的に屈曲）した流路部位を有し、当該屈曲した流路部位は、これより上流の希釈流路の軸線方向ないしその延長方向をＸ方向と、このＸ方向と垂直に交差する希釈流路の幅方向をＹ方向とし、これより上流の希釈流路の流路幅をｙ０とした場合に、Ｙ方向において対向する希釈流路の両側壁面より交互に、流路中心側に向かって、略Ｙ方向（略＋Ｙ方向、略－Ｙ方向）に、１／２ｙ０以上１ｙ０未満の一定高さｈ１、ｈ２．．．を有し、かつＸ方向に一定幅ｘ１、ｘ２．．．を有して突出し、希釈流路の流路幅を規制する構造子が、一定間隔ｄ１、ｄ２．．．をもって少なくとも２つ以上設けられていることで形成されている流路構造体である。 The microchannel structure described in Patent Document 2 has a first introduction path for introducing a first fluid and a second introduction path for introducing a second fluid, which are independent of each other on the upstream side. , Each of which has a constant length and merges to form one dilution channel toward the downstream side thereof, and the dilution channel is bent (for example, two-dimensionally bent) at least in a part thereof. The bent flow path portion has a flow path portion, and the axial direction of the dilution flow path upstream from this or the extension direction thereof is the X direction, and the width direction of the dilution flow path perpendicular to the X direction is Y. When the flow path width of the dilution flow path upstream from this is set to y0, the direction is approximately Y (approximately + Y) toward the center of the flow path alternately from both side walls of the dilution flow paths facing each other in the Y direction. Constant heights h1 and h2 of 1 / 2y0 or more and less than 1y0 in the direction (approximately −Y direction). .. .. And has a constant width x1 and x2 in the X direction. .. .. The structure that regulates the flow path width of the dilution flow path is formed at regular intervals d1, d2. .. .. It is a flow path structure formed by providing at least two of them.
　前記流路構造体において、流路幅ｙ０は、好ましくは２０～１０００μｍであり、より好ましくは１００～４００μｍであり、さらに好ましくは１５０～３００μｍの範囲である。各構造子の幅ｘ１，ｘ２．．．は、好ましくは２０～１０００μｍであり、より好ましくは５０～３００μｍであり、さらに好ましくは７０～２００μｍの範囲である。各構造子間の間隔ｄ１，ｄ２．．．は、好ましくは２０～１０００μｍであり、より好ましくは５０～４００μｍであり、さらに好ましくは７０～２００μｍの範囲である。さらに各構造子の高さｈ１，ｈ２．．．は、好ましくは１／２ｙ０以上、３／４ｙ０以下である。構造子は、好ましくは１０～１００個、より好ましくは１０～５０個、さらに好ましくは１５～３０個の範囲設けられている。 In the flow path structure, the flow path width y0 is preferably 20 to 1000 μm, more preferably 100 to 400 μm, and further preferably 150 to 300 μm. Width x1, x2 of each structure. .. .. Is preferably 20 to 1000 μm, more preferably 50 to 300 μm, and even more preferably 70 to 200 μm. Spacing between each structure d1, d2. .. .. Is preferably 20 to 1000 μm, more preferably 50 to 400 μm, and even more preferably 70 to 200 μm. Further, the heights of each structure h1, h2. .. .. Is preferably 1 / 2y0 or more and 3/4y0 or less. The structure is preferably provided in the range of 10 to 100, more preferably 10 to 50, and even more preferably 15 to 30.
　前記流路構造体において、脂質相としてアルコール溶液を第１導入路に導入し、水相として水系溶媒を第２導入路に導入する。第１導入路と第２導入路との合流点より最初の構造子の上流側端部までの距離は、適宜設定することができるが、この間を流れる設定速度の希釈流体が０．１秒以下で通過するように、希釈流体の設定速度に応じて規定されていることが好ましい。難水溶性化合物をアルコール溶液に溶解させるために加温する場合には、流路を加温してもよい。 In the flow path structure, an alcohol solution is introduced into the first introduction path as a lipid phase, and an aqueous solvent is introduced into the second introduction path as an aqueous phase. The distance from the confluence of the first introduction path and the second introduction path to the upstream end of the first structure is appropriately set, but the diluted fluid at the set speed flowing between them is 0.1 seconds or less. It is preferable that it is specified according to the set speed of the diluting fluid so as to pass through. When heating the poorly water-soluble compound to dissolve it in the alcohol solution, the flow path may be heated.
　マイクロ流路へのアルコール溶液及び水系溶媒の各供給量は、マイクロ流路の出口から回収される分散体のアルコール濃度が４０％以下になる量に制御することが、所望の粒子径及び分散度を有する脂質膜構造体を分散質として含有する分散体を得ることができるので好ましい。好ましくはマイクロ流路の出口から回収される分散体のアルコール濃度が５～３５％の範囲、より好ましくは１０～３０％の範囲になる量に制御する。マイクロ流路構造体での操作は、溶媒の沸点も考慮しつつ、例えば、０～７０℃の範囲の温度で実施することができる。マイクロ流路内でアルコール溶液を水系溶媒で希釈して、難水溶性化合物を含有する脂質膜構造体を分散質として含有する分散体をマイクロ流路の出口から回収する。 The amount of each supply of the alcohol solution and the aqueous solvent to the microchannel is controlled so that the alcohol concentration of the dispersion recovered from the outlet of the microchannel is 40% or less, which is the desired particle size and dispersion degree. It is preferable because it is possible to obtain a dispersion containing the lipid film structure having the above as a dispersoid. The alcohol concentration of the dispersion recovered from the outlet of the microchannel is preferably controlled to be in the range of 5 to 35%, more preferably in the range of 10 to 30%. The operation in the microchannel structure can be carried out at a temperature in the range of 0 to 70 ° C., for example, while considering the boiling point of the solvent. The alcohol solution is diluted with an aqueous solvent in the microchannel, and the dispersion containing the lipid membrane structure containing the poorly water-soluble compound as a dispersoid is recovered from the outlet of the microchannel.
　マイクロ流路の出口から回収した分散体からアルコールを除去する工程をさらに含むことができる。分散体からのアルコール除去は、例えば、透析、蒸留などで行うことができる。透析は、例えば、０℃～室温で行うことができる。 A step of removing alcohol from the dispersion recovered from the outlet of the microchannel can be further included. Alcohol removal from the dispersion can be performed, for example, by dialysis, distillation, or the like. Dialysis can be performed, for example, from 0 ° C. to room temperature.
　分散体またはアルコールを除去した分散体は、さらに濃縮工程に付すこともできる。濃縮工程は、例えば、限外ろ過、遠心分離、溶媒（水）の蒸発や透析であることができる。限外ろ過は、例えば、限外ろ過膜を用いて行うことができる。限外ろ過膜は、所定の名目分子量カットオフ（ＮＭＣＯ）を有し、所定のＮＭＣＯは、特に限定されないが、５０ｋＤａ～２００ｋＤａの範囲のいずれかのＮＭＣＯであり得、取得する分散体に含まれる脂質膜構造体の粒径に合わせて適宜選択することができる。 The dispersion or the dispersion from which the alcohol has been removed can be further subjected to a concentration step. The concentration step can be, for example, ultrafiltration, centrifugation, evaporation of solvent (water) or dialysis. Ultrafiltration can be performed using, for example, an ultrafiltration membrane. The ultrafiltration membrane has a predetermined nominal molecular weight cutoff (NMCO), and the predetermined NMCO can be any NMCO in the range of 50 kDa to 200 kDa, and is included in the dispersion to be obtained. It can be appropriately selected according to the particle size of the lipid membrane structure.
　本発明は、難水溶性化合物を含有し、動的光散乱（ＤＬＳ）法で測定した平均粒子径が６０ｎｍ以下である脂質膜構造体を分散質として分散媒中に含有する分散体であって、前記脂質膜構造体の脂質膜は、リン脂質及び脂質修飾ポリエチレングリコールを含有する、前記分散体である。本発明の分散体は、前記脂質膜構造体の脂質膜が膜透過性ペプチドを含むことができる。これらの分散体は上記本発明の製造方法により調製することができる。但し、分散体の脂質膜構造体の脂質膜が膜透過性ペプチドを含まない場合には、リン脂質等を含有するアルコール溶液（マイクロ流路に導入する脂質相）として膜透過性ペプチドを含まないアルコール溶液を用いる。 The present invention is a dispersion containing a poorly water-soluble compound and containing a lipid film structure having an average particle diameter of 60 nm or less measured by a dynamic light scattering (DLS) method as a dispersant in a dispersion medium. The lipid membrane of the lipid membrane structure is the dispersion containing phospholipids and lipid-modified polyethylene glycol. In the dispersion of the present invention, the lipid membrane of the lipid membrane structure can contain a membrane-permeable peptide. These dispersions can be prepared by the above-mentioned production method of the present invention. However, when the lipid membrane of the lipid membrane structure of the dispersion does not contain a membrane-permeable peptide, the membrane-permeable peptide is not contained as an alcohol solution (lipid phase introduced into the microchannel) containing phospholipids and the like. Use an alcohol solution.
　脂質膜構造体の脂質膜を構成するリン脂質、膜透過性ペプチド及び脂質修飾ポリエチレングリコールの種類については、前記製造方法に関する記載を参照する。 For the types of phospholipids, membrane-permeable peptides, and lipid-modified polyethylene glycols that make up the lipid membrane of the lipid membrane structure, refer to the description of the above-mentioned production method.
　特に、特許文献２及び非特許文献５に記載のマイクロ流路構造体を用い、ＤＬＳ法で測定した平均粒子径が６０ｎｍ以下である脂質膜構造体を分散質として分散媒中に含有する分散体が得られる。 In particular, a dispersion containing a lipid film structure having an average particle diameter of 60 nm or less measured by a DLS method as a dispersoid in a dispersion medium using the microchannel structures described in Patent Document 2 and Non-Patent Document 5. Is obtained.
　リン脂質は、ジオレイルホスファチジルエタノールアミンとホスファチジン酸及び／又はスフィンゴミエリンであることが、ミトコンドリア内への目的物質である難水溶性化合物の効果的な送達のために好ましい。 Phospholipids are preferably dioleylphosphatidylethanolamine and phosphatidic acid and / or sphingomyelin for effective delivery of the poorly water-soluble compound which is the target substance into the mitochondria.
　脂質膜構造体（または脂質ナノ粒子）が膜透過性ペプチドを含有する場合の膜透過性ペプチドの含有量は、脂質膜の総量に対して、例えば、５～２０モル％、好ましくは１０～１５モル％の範囲である。 When the lipid membrane structure (or lipid nanoparticles) contains a membrane-permeable peptide, the content of the membrane-permeable peptide is, for example, 5 to 20 mol%, preferably 10 to 15%, based on the total amount of the lipid membrane. It is in the range of mol%.
　脂質膜構造体（または脂質ナノ粒子）における脂質修飾ＰＥＧの含有量は、脂質膜の総量に対して、例えば、１～１０モル％、好ましくは３～５モル％の範囲である。 The content of lipid-modified PEG in the lipid membrane structure (or lipid nanoparticles) is, for example, in the range of 1 to 10 mol%, preferably 3 to 5 mol%, based on the total amount of the lipid membrane.
　脂質膜構造体（または脂質ナノ粒子）における難水溶性化合物の量は、脂質膜の総量に対して、例えば、１０～４０モル％、好ましくは１５～３０モル％の範囲、２０～２５モル％の範囲である。 The amount of the poorly water-soluble compound in the lipid membrane structure (or lipid nanoparticles) is, for example, in the range of 10 to 40 mol%, preferably 15 to 30 mol%, or 20 to 25 mol%, based on the total amount of the lipid membrane. Is the range of.
　但し、いずれもこれらの範囲に制限される意図ではない。 However, neither is intended to be limited to these ranges.
　本発明の分散体は、ＤＬＳ法で測定した脂質膜構造体（または脂質ナノ粒子）の多分散性指数（ＰＤＩまたはＰｄＩ）が、好ましくは０．３以下であり、好ましくは０．２５以下の範囲である。多分散性指数（ＰＤＩ）は小さいほど好ましく、特に限定されないが、実質的には下限値は０．１程度であり得る。 In the dispersion of the present invention, the polydispersity index (PDI or PdI) of the lipid membrane structure (or lipid nanoparticles) measured by the DLS method is preferably 0.3 or less, preferably 0.25 or less. The range. The smaller the polydispersity index (PDI) is, the more preferable it is, and the lower limit value may be substantially 0.1, although it is not particularly limited.
　本発明の分散体は、脂質膜構造体（または脂質ナノ粒子）のゼータ電位が、特に限定されないが例えば、１０ｍＶ以上、１１ｍＶ以上、１２ｍＶ以上、１３ｍＶ以上、１４ｍＶ以上、または好ましくは１５ｍＶ以上の範囲である。ゼータ電位は、大きい程好ましく、特に限定されないが、実質的には上限値は５０ｍＶ程度であり得る。本発明の分散体の脂質膜構造体（または脂質ナノ粒子）のゼータ電位はゼータサイザーナノＺＳ（Ｍａｌｖｅｒｎ社、Ｗｏｒｃｅｓｔｅｒｓｈｉｒｅ、ＵＫ）によって、レーザードップラー式電気泳動法を用いて測定することができる。 In the dispersion of the present invention, the zeta potential of the lipid membrane structure (or lipid nanoparticles) is not particularly limited, but is, for example, in the range of 10 mV or more, 11 mV or more, 12 mV or more, 13 mV or more, 14 mV or more, or preferably 15 mV or more. Is. The larger the zeta potential is, the more preferable it is, and the upper limit value can be substantially about 50 mV, although it is not particularly limited. The zeta potential of the lipid membrane structure (or lipid nanoparticles) of the dispersion of the present invention can be measured by the Zetasizer Nano ZS (Malvern, Worcestershire, UK) using laser Doppler electrophoresis.
　本発明の分散体は、分散媒が水系溶媒である。水系溶媒の例は、前掲の通りである。本発明の分散体は、ある態様では、分散媒がアルコールを含有しない、または分散媒からアルコールが除去されている。 The dispersion medium of the dispersion of the present invention is an aqueous solvent. Examples of aqueous solvents are as described above. In some aspects of the dispersion of the present invention, the dispersion medium does not contain alcohol, or alcohol is removed from the dispersion medium.
　本発明の分散体は、難水溶性化合物を細胞内、および細胞内のミトコンドリアに移送するために用いられる。 The dispersion of the present invention is used to transfer a poorly water-soluble compound to intracellular and intracellular mitochondria.
　本発明の分散体は、特に限定されないが例えば、リン脂質が、ジオレイルホスファチジルエタノールアミンとホスファチジン酸及び／又はスフィンゴミエリンである、分散体であり、難水溶性化合物としてＣｏＱ１０を脂質膜の総量に対して、１０～４０モル％の範囲で含有し、脂質膜構造体（または脂質ナノ粒子）のＤＬＳ法で測定した６０ｎｍ以下、好ましくは５５ｎｍ以下、より好ましくは５０ｎｍ以下の脂質膜構造体（または脂質ナノ粒子）を分散質として含有する。本発明の分散体は、ある態様では、ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンとを含む脂質膜構造体（または脂質ナノ粒子）を含み、ＣｏＱ１０を脂質膜の総量に対して、１０～４０モル％の範囲で含有し、ＤＬＳ法で測定した粒径において６０ｎｍ以下、好ましくは５５ｎｍ以下、より好ましくは５０ｎｍ以下の脂質膜構造体（または脂質ナノ粒子）を含有し、かつ、ＤＬＳ法で測定したときに０．３未満のＰＤＩをさらに有し得る。ここで、脂質膜構造体（または脂質ナノ粒子）は分散質である。平均粒子径は、細胞への取り込み効率などの観点からはある程度小さい方が好ましく、特に限定されないが、下限値は１０ｎｍ程度であり得、好ましくは２０ｎｍ程度であり得る。ある態様では、本発明の分散体は、ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンとを含む脂質膜構造体（または脂質ナノ粒子）を含み、脂質膜構造体（または脂質ナノ粒子）はＣｏＱ１０を脂質膜の総量に対して、１０～４０モル％の範囲で含有し、脂質膜構造体（または脂質ナノ粒子）はＤＬＳ法で測定した平均粒径が５０ｎｍ～６０ｎｍであり、かつ、０．３未満のＰＤＩを有し得る。ここで、脂質膜構造体（または脂質ナノ粒子）は分散質である。 The dispersion of the present invention is not particularly limited, but is, for example, a dispersion in which the phospholipids are dioleylphosphatidylethanolamine, phosphatidylic acid and / or sphingomyelin, and CoQ 10 as a poorly water-soluble compound is used as the total amount of the lipid membrane. A lipid film structure containing 10 to 40 mol% of the lipid film structure (or lipid nanoparticles) measured by the DLS method and having a lipid film structure of 60 nm or less, preferably 55 nm or less, more preferably 50 nm or less. Or lipid nanoparticles) as a dispersoid. In some embodiments, the dispersion of the present invention comprises a lipid membrane structure (or lipid nanoparticles) comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin and dioleylphosphatidylethanolamine. , CoQ 10 is contained in the range of 10 to 40 mol% with respect to the total amount of the lipid membrane, and the lipid membrane structure having a particle size measured by the DLS method of 60 nm or less, preferably 55 nm or less, more preferably 50 nm or less ( Or lipid nanoparticles) and may further have a PDI of less than 0.3 when measured by the DLS method. Here, the lipid membrane structure (or lipid nanoparticles) is a dispersoid. The average particle size is preferably small to some extent from the viewpoint of efficiency of uptake into cells, and is not particularly limited, but the lower limit value can be about 10 nm, preferably about 20 nm. In some embodiments, the dispersion of the present invention comprises a lipid membrane structure (or lipid nanoparticles) comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin and dioleylphosphatidylethanolamine. , The lipid membrane structure (or lipid nanoparticles) contained CoQ 10 in the range of 10 to 40 mol% with respect to the total amount of the lipid membrane, and the lipid membrane structure (or lipid nanoparticles) was measured by the DLS method. It can have an average particle size of 50 nm to 60 nm and a PDI of less than 0.3. Here, the lipid membrane structure (or lipid nanoparticles) is a dispersoid.
　本発明のある態様では、難水溶性化合物は、脂溶性化合物であり得る。本発明のある態様では、難水溶性化合物は、アルコール（例えば、エタノール）に溶ける。本発明のある態様では、難水溶性化合物は、脂溶性のＢＣＳクラス４の化合物であり得る。本発明のある態様では、難水溶性化合物は、エタノールに溶けるＢＣＳクラス４の化合物であり得る。難水溶性化合物のアルコールへの溶解性を向上または改善するために、加熱条件下で当該化合物をアルコールに溶解させることをさらに含んでいてもよい。好ましい態様では、ＣｏＱ１０は、加熱条件下（例えば、５０℃）でエタノールに溶解させて脂質膜構造体（または脂質ナノ粒子）の調製に用いることができ、溶解させた状態で脂質膜構造体（または脂質ナノ粒子）に内包させることで、天然に近い状態で膜に取り込ませることができ、それ故に、細胞内に送達されたＣｏＱ１０が送達された場所において本来の機能性を十分に発揮し得る。 In some aspects of the invention, the poorly water-soluble compound can be a fat-soluble compound. In some aspects of the invention, the poorly water soluble compound is soluble in alcohol (eg, ethanol). In some aspects of the invention, the poorly water-soluble compound can be a fat-soluble BCS class 4 compound. In some aspects of the invention, the poorly water soluble compound can be a BCS class 4 compound that is soluble in ethanol. In order to improve or improve the solubility of the poorly water-soluble compound in alcohol, it may further include dissolving the compound in alcohol under heating conditions. In a preferred embodiment, CoQ 10 can be dissolved in ethanol under heating conditions (eg, 50 ° C.) for use in the preparation of lipid membrane structures (or lipid nanoparticles), and in the dissolved state the lipid membrane structures. By encapsulation in (or lipid nanoparticles), it can be incorporated into the membrane in a state close to nature, and therefore, the original functionality is fully exhibited at the place where CoQ 10 delivered intracellularly is delivered. Can be.
　本発明によれば、本発明の難水溶性化合物を含む分散体を含む医薬製剤が提供される。本発明によれば、ＣｏＱ１０を含む本発明の分散体を含む医薬製剤が提供される。 According to the present invention, a pharmaceutical preparation containing a dispersion containing the poorly water-soluble compound of the present invention is provided. According to the present invention, a pharmaceutical preparation containing the dispersion of the present invention containing CoQ 10 is provided.
　本発明において、難水溶性化合物がＣｏＱ１０である組成物は、例えば、呼吸鎖複合体Ｉに機能不全を有する対象に投与され得る。本発明において、難水溶性化合物がＣｏＱ１０である組成物は、呼吸鎖複合体（例えば、呼吸鎖複合体Ｉ、ＩＩ、ＩＩＩ、およびＩＶのいずれか１以上）に機能不全を有する対象などのミトコンドリア病に罹患した対象において、細胞のＡＴＰ産生を向上させることに用いられ得る。呼吸鎖複合体Ｉに機能不全を有する対象は、特に限定されないが例えば、ミトコンドリア脳筋症（ＭＥＬＡＳ）に罹患した対象、Ｌｅｉｇｈ脳症に罹患した対象、レーベル遺伝性視神経症（ＬＨＯＮ）に罹患した対象が挙げられる。また、本発明において、難水溶性化合物がＣｏＱ１０である組成物は、例えば、ＣｏＱ１０欠乏症に罹患した対象が挙げられる。ここで、対象は、動物、特に哺乳動物、特に霊長類、特に好ましくはヒトであり得る。 In the present invention, the composition in which the poorly water-soluble compound is CoQ 10 can be administered to, for example, a subject having a dysfunction in the respiratory chain complex I. In the present invention, the composition in which the poorly water-soluble compound is CoQ 10 is a subject having a dysfunction in the respiratory chain complex (for example, any one or more of the respiratory chain complexes I, II, III, and IV). It can be used to improve ATP production in cells in subjects suffering from mitochondrial disease. Subjects having dysfunction in respiratory chain complex I are not particularly limited, but are, for example, subjects suffering from mitochondrial encephalomyopathy (MELAS), subjects suffering from Leigh encephalopathy, and subjects suffering from Leber's hereditary optic neuropathy (LHON). Can be mentioned. Further, in the present invention, the composition in which the poorly water-soluble compound is CoQ 10 includes, for example, a subject suffering from CoQ 10 deficiency. Here, the subject can be an animal, particularly a mammal, particularly a primate, particularly preferably a human.
　本発明によれば、対象に難水溶性化合物を投与する方法であって、対象に本発明の難水溶性化合物を含む分散体を投与することを含む、方法が提供される。
　本発明によれば、対象の体内の細胞内に難水溶性化合物を送達する方法であって、対象に本発明の難水溶性化合物を含む分散体を投与することを含む、方法が提供される。
　本発明によれば、対象の体内の細胞内のミトコンドリアに難水溶性化合物を送達する方法であって、対象に本発明の難水溶性化合物を含む分散体を投与することを含む、方法が提供される。
　本発明によれば、ミトコンドリア病に罹患した対象において、ミトコンドリア病を処置する方法であって、対象に本発明の難水溶性化合物を含む分散体を投与することを含む、方法が提供される。
　本発明によれば、ミトコンドリア病に罹患した対象において、ミトコンドリア病を処置する方法であって、対象にＣｏＱ１０を含む本発明の分散体を投与することを含む、方法が提供される。 According to the present invention, there is provided a method for administering a poorly water-soluble compound to a subject, which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention.
According to the present invention, there is provided a method for delivering a poorly water-soluble compound into cells in a subject's body, which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention. ..
According to the present invention, there is provided a method for delivering a poorly water-soluble compound to mitochondria in a cell in a subject body, which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention. Will be done.
According to the present invention, there is provided a method for treating mitochondrial disease in a subject suffering from mitochondrial disease, which comprises administering to the subject a dispersion containing the poorly water-soluble compound of the present invention.
According to the present invention, there is provided a method for treating mitochondrial disease in a subject suffering from mitochondrial disease, which comprises administering the dispersion of the present invention containing CoQ 10 to the subject.
　本発明によれば、難水溶性化合物を含む本発明の分散体を含む医薬製剤の製造における、難水溶性化合物の使用が提供される。本発明によれば、ＣｏＱ１０を含む本発明の分散体分散体を含む医薬製剤の製造における、ＣｏＱ１０の使用が提供される。　本発明によれば、難水溶性化合物を含む本発明の分散体を含む医薬製剤の製造における、リン脂質および脂質修飾非電荷親水性ポリマーの使用が提供される。本発明によれば、ＣｏＱ１０を含む本発明の分散体分散体を含む医薬製剤の製造における、リン脂質および脂質修飾非電荷親水性ポリマーの使用が提供される。
　本発明によれば、難水溶性化合物を含む本発明の分散体を含む医薬製剤の製造における、難水溶性化合物、リン脂質および脂質修飾非電荷親水性ポリマーの使用が提供される。本発明によれば、ＣｏＱ１０を含む本発明の分散体分散体を含む医薬製剤の製造における、ＣｏＱ１０、リン脂質および脂質修飾非電荷親水性ポリマーの使用が提供される。 INDUSTRIAL APPLICABILITY According to the present invention, the use of a poorly water-soluble compound in the production of a pharmaceutical preparation containing the dispersion of the present invention containing the poorly water-soluble compound is provided. According to the present invention, in the manufacture of a pharmaceutical formulation comprising a dispersion dispersion of the present invention containing CoQ 10, the use of CoQ 10 is provided. INDUSTRIAL APPLICABILITY The present invention provides the use of phospholipids and lipid-modified uncharged hydrophilic polymers in the manufacture of pharmaceutical formulations comprising the dispersions of the present invention containing poorly water soluble compounds. The present invention provides the use of phospholipids and lipid-modified uncharged hydrophilic polymers in the manufacture of pharmaceutical formulations comprising the dispersion dispersions of the present invention containing CoQ 10 .
INDUSTRIAL APPLICABILITY The present invention provides the use of poorly water-soluble compounds, phospholipids and lipid-modified uncharged hydrophilic polymers in the manufacture of pharmaceutical formulations comprising the dispersions of the present invention containing poorly water-soluble compounds. According to the present invention, in the manufacture of a pharmaceutical formulation comprising a dispersion dispersion of the present invention containing CoQ 10, CoQ 10, the use of phospholipids and lipid-modified uncharged hydrophilic polymer.
　本発明の分散体は、医薬製剤化され得る。本発明の分散体を含む医薬製剤は、薬学的に許容可能な賦形剤をさらに含んでいてもよい。賦形剤としては、特に限定されないが、緩衝剤、等張化剤、薬学的に許容可能な塩、分散剤、抗酸化剤、保存剤、および無痛化剤が挙げられる。 The dispersion of the present invention can be made into a pharmaceutical formulation. Pharmaceutical formulations containing the dispersions of the invention may further contain pharmaceutically acceptable excipients. Excipients include, but are not limited to, buffering agents, tonicity agents, pharmaceutically acceptable salts, dispersants, antioxidants, preservatives, and soothing agents.
　本発明の分散体は、化粧品またはサプリメントとして調製され得る。したがって、本発明によれば、本発明の分散体を含む化粧品およびサプリメントが提供され得る。 The dispersion of the present invention can be prepared as a cosmetic or supplement. Therefore, according to the present invention, cosmetics and supplements containing the dispersion of the present invention may be provided.
　以下、本発明を実施例に基づいて更に詳細に説明する。但し、実施例は本発明の例示であって、本発明は実施例に限定される意図ではない。 Hereinafter, the present invention will be described in more detail based on examples. However, the examples are examples of the present invention, and the present invention is not intended to be limited to the examples.
＜準備するもの＞
□脂質溶液（７．５　ｍＭ　ＤＯＰＥ、７．５　ｍＭ　ＳＭ、７．５　ｍＭ　ＤＭＧ－ＰＥＧ　２０００、および１．５　ｍＭ　ＣｏＱ１０を含むエタノール溶液）
ＤＯＰＥ：　１，２－ジオレオイル－ｓｎ－グリセロ－３－ホスホエタノールアミン
ＳＭ：　スフィンゴミエリン
ＤＭＧ－ＰＥＧ　２０００：　１，２－ジミリストイル－ｓｎ－グリセロール，　メトキシポリエチレングリコール　２０００□ＥｔＯＨ　
□ＰＢＳ（－）（ＰＢＳタブレット、ＴａＫａＲａ、Ｔ９００）　
□２０　ｍｇ／ｍＬ　ＳＴＲ－Ｒ８を含むエタノール溶液
ＳＴＲ－Ｒ８：　ステアリル化Ｒ８
□透析膜Ｓｐｅｃｔｒａ／Ｐｏｒ　４　ｄｉａｌｙｓｉｓ　ｍｅｍｂｒａｎｅ　（ＭＷＣＯ　１２ｋ－１４ｋ、　Ｓｐｅｃｔｒａｍ　Ｌａｂｐｒａｔｏｒｉｅｓ）
□１　ｍＬ、　２．５　ｍＬガラスシリンジ（ＨＡＭＩＬＴＯＮ）
□シリンジポンプＳｔａｎｄａｒｄ　ｉｎｆｕｓｉｏｎ　Ｏｎｌｙ　Ｐｕｍｐ　１１　Ｅｌｉｔｅ（ＨＡＲＶＡＲＤ　ＡＰＰＡＲＡＴＵＳ）　□透析膜クリップＳｐｅｃｔｒａ／Ｐｏｒ　Ｃｌｏｓｕｒｅｓ <What to prepare>
□ Lipid solution (ethanol solution containing 7.5 mM DOPE, 7.5 mM SM, 7.5 mM DMG-PEG 2000, and 1.5 mM CoQ 10 )
DOPE: 1,2-diore oil-sn-glycero-3-phosphoethanolamine SM: sphingomyelin DMG-PEG 2000: 1,2-dimylistyl-sn-glycerol, methoxypolyethylene glycol 2000 □ EtOH
□ PBS (-) (PBS tablet, TakaRa, T900)
□ Ethanol solution containing 20 mg / mL STR-R8 STR-R8: Stearylinated R8
□ Dialysis Membrane Spectra / Por 4 dialysis membrane (MWCO 12k-14k, Spectram Labplatries)
□ 1 mL, 2.5 mL glass syringe (HAMILTON)
□ Syringe Pump Standard Infusion Only Pump 11 Elite (HarVARD APPARATUS) □ Dialysis Membrane Clip Spectra / Por Closures
＜手順＞
１．　脂質溶液、ＰＢＳ（－）をすべて室温に戻す。ＣｏＱ１０溶液は５０℃２分間加温　（袋に入れて）又は、ソニケーションで溶解させる。
２．　透析用ＰＢＳ（－）５００　ｍＬを作製し、撹拌させながら２５℃で保存する。
３．　ビーカーにＤＤＷ３００～４００　ｍＬを入れ、透析膜Ｓｐｅｃｔｒａ／Ｐｏｒ　４　ｄｉａｌｙｓｉｓ　ｍｅｍｂｒａｎｅ　（ＭＷＣＯ　１２ｋ－１４ｋ）を適当な長さに切り、膜がくっつかないように激しめに撹拌し、水和させる（３０　分以上）。
４．　エッペンドルフチューブに以下の量の各溶液を加え、脂質溶液（脂質相）を調製する。 <Procedure>
1. 1. Bring all lipid solutions and PBS (-) to room temperature. The CoQ10 solution is heated at 50 ° C. for 2 minutes (in a bag) or dissolved by sonication.
2. Make 500 mL of PBS (-) for dialysis and store at 25 ° C. with stirring.
3. 3. Put 300-400 mL of DDW in a beaker, cut the dialysis membrane Spectra / Por 4 dialysis membrane (MWCO 12k-14k) to an appropriate length, stir vigorously so that the membrane does not stick, and hydrate (30 minutes or more). ).
4. Add the following amounts of each solution to the Eppendorf tube to prepare a lipid solution (lipid phase).
Figure JPOXMLDOC01-appb-C000001
Figure JPOXMLDOC01-appb-C000001
５．　ＰＢＳ（水相）も必要量用意する。
６．　バッフルミキサー内臓マイクロ流路にキャップ、コネクター、シリンジ接合部を装着する（向きに注意する）。
７．　脂質溶液を１　ｍＬ、ＰＢＳ（－）を２．５　ｍＬガラスシリンジにそれぞれ充填し、シリンジをシリンジポンプに装着する。
※使用するシリンジと機器の径があっているか確認する。
８．　マイクロ流路とポンプを接続し、脂質相、水相をそれぞれの流速で流し、空気が追い出されるのを確認する。混合後、はじめは廃液に回収、キムワイプで先端を拭いてからエッペンドルフチューブに適当な容量（２５０μＬ）を回収する。
※総流速５００～１０００μＬ／ｍｉｎ
※ＦＲＲ（水相／脂質相μＬ／ｍｉｎ）　総流速５００　μＬ／ｍｉｎの場合
ｅｘ）・ＦＲＲ１（５０％希釈）：　２５０／２５０　・ＦＲＲ３（２５％希釈）：　３７５／１２５　・ＦＲＲ４（２０％希釈）：４００／１００　・ＦＲＲ９（１０％希釈）：　４５０／５０
※連続で使用する場合、シリンジはＥｔＯＨで洗浄し、流路は空気が抜けるのを確認し、なじませてから回収する。 5. Prepare the required amount of PBS (aqueous phase).
6. Attach the cap, connector, and syringe joint to the micro flow path built into the baffle mixer (note the orientation).
7. Fill a glass syringe with 1 mL of lipid solution and 2.5 mL of PBS (-), respectively, and attach the syringe to the syringe pump.
* Check that the diameter of the syringe used and the device match.
8. Connect the micro flow path and the pump, let the lipid phase and the aqueous phase flow at their respective flow velocities, and confirm that the air is expelled. After mixing, first collect in waste liquid, wipe the tip with a Kimwipe, and then collect an appropriate volume (250 μL) in an Eppendorf tube.
* Total flow velocity 500-1000 μL / min
* FRR (aqueous phase / lipid phase μL / min) when the total flow velocity is 500 μL / min ex) ・ FRR1 (50% dilution): 250/250 ・ FRR3 (25% dilution): 375/125 ・ FRR4 (20% dilution) ): 400/100 ・ FRR9 (10% dilution): 450/50
* When using continuously, wash the syringe with EtOH, check that air is released from the flow path, and let it blend in before collecting.
９．　透析膜をビーカーから取り出し、水気をとり除き、白いクリップで片方を閉じ、膜をよじって中にＬＰ溶液（すなわち、マイクロ流路から得られた分散体）を入れ、オレンジのクリップでもう片方を閉じて浮きを付け、ＰＢＳ（－）を透析外液として用いて、冷温室で２　時間以上透析する。その後、透析膜から溶液を回収する。
１０．　ＥｔＯＨでシリンジは５回（はじめの２回はシリンジの先端も洗浄）、コネクターは３回洗浄する。流路は５０μＬ／ｍｉｎで１０　分間洗浄する。流路は洗浄後、シリンジを用いて空気で２回置換する。 9. Remove the dialysis membrane from the beaker, drain it, close one with a white clip, twist the membrane into the LP solution (ie the dispersion obtained from the microchannel), and clip the other with an orange clip. Close and float and dialyze in a cold greenhouse for at least 2 hours using PBS (-) as an external dialysis solution. The solution is then recovered from the dialysis membrane.
10. Clean the syringe 5 times with EtOH (the first 2 times also clean the tip of the syringe) and the connector 3 times. The flow path is washed at 50 μL / min for 10 minutes. After cleaning the flow path, replace it with air twice using a syringe.
実施例１
１－１．　マイクロ流路を用いたＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの調製法
　図１のマイクロ流路デバイスを用いて粒子調製を行った。シリンジポンプは、ＨＡＲＶＡＲＤ　ＡＰＰＡＲＡＴＵＳ社のＳｔａｎｄａｒｄ　ｉｎｆｕｓｉｏｎ　Ｏｎｌｙ　Ｐｕｍｐ　１１　Ｅｌｉｔｅを用いた。シリンジは、ＨＡＭＩＬＴＯＮ社のガラスシリンジを１　ｍＬ（脂質相）と２．５　ｍＬ（水相）を用いた。マイクロ流路を用いた調製では、（ｉ）脂質相の濃度、（ｉｉ）水相の種類、（ｉｉｉ）総流速、（ｉｖ）流速比（水相／脂質相の流速の比率＝ＥｔＯＨ希釈濃度に相当）のより多彩な粒子設計が可能となる。 Example 1
1-1. Preparation method of CoQ 10- MITO-Porter using microchannel The particles were prepared using the microchannel device shown in FIG. As the syringe pump, a Standard Infusion Only Pump 11 Elite manufactured by HARVARD APPARATUS was used. As the syringe, 1 mL (lipid phase) and 2.5 mL (aqueous phase) of a glass syringe manufactured by HAMILTON were used. In the preparation using the microchannel, (i) the concentration of the lipid phase, (ii) the type of the aqueous phase, (iii) the total flow velocity, and (iv) the flow velocity ratio (the ratio of the flow velocity of the aqueous phase / the lipid phase = EtOH dilution concentration). (Equivalent to) enables a wider variety of particle designs.
　（ｉ）脂質相
　７．５　ｍＭ　ＤＯＰＥ（ＥｔＯＨ）、７．５　ｍＭ　ＳＭ（ＥｔＯＨ）、および７．５　ｍＭ　ＤＭＧ－ＰＥＧ　２０００（ＥｔＯＨ）を含むエタノール溶液を調製し、室温とした。エッペンドルフチューブに上図の体積比となるように脂質材料を含有したエタノール懸濁液を準備し、（ｉ）脂質相とした。さらに脂質相には脂質濃度に対して１０　ｍｏｌ％修飾となるように２０　ｍｇ／ｍＬのＳＴＲ－Ｒ８を１２．６μＬ含有させた。従来法ではＣｏＱ１０の沈殿が確認されることもあったので、濃度を薄くした１．５　ｍＭ　ＣｏＱ１０（ＥｔＯＨ）とし、さらに加温（５０℃、２分間）し、超音波処理して希薄溶液を調製した。調製した１．５　ｍＭＣｏＱ１０（ＥｔＯＨ）は室温下で沈殿することはなかった。一方で、５　ｍＭＣｏＱ１０（ＥｔＯＨ）は室温下では黄色い濁りを有する懸濁液であった。
　（ｉｉ）水相は従来法の希釈溶媒であるＰＢＳ（－）を用いた。
　（ｉｉｉ）総流速は５００μＬ／ｍｉｎとした。 (I) Lipid phase An ethanol solution containing 7.5 mM DOPE (EtOH), 7.5 mM SM (EtOH), and 7.5 mM DMG-PEG 2000 (EtOH) was prepared and brought to room temperature. An ethanol suspension containing a lipid material was prepared in an Eppendorf tube so as to have the volume ratio shown in the above figure, and was used as (i) a lipid phase. Further, the lipid phase contained 12.6 μL of 20 mg / mL STR-R8 so as to be modified by 10 mol% with respect to the lipid concentration. Precipitation of CoQ 10 was sometimes confirmed by the conventional method, so the concentration was reduced to 1.5 mM CoQ 10 (EtOH), further heated (50 ° C for 2 minutes), and sonicated to dilute it. The solution was prepared. The prepared 1.5 mM CoQ 10 (EtOH) did not precipitate at room temperature. On the other hand, 5 mM CoQ 10 (EtOH) was a suspension having a yellow turbidity at room temperature.
(Ii) As the aqueous phase, PBS (−), which is a conventional diluting solvent, was used.
(Iii) The total flow velocity was set to 500 μL / min.
　（ｉｖ）流速比
　流速比は、エタノール希釈濃度が１０％、２０％、３０％、または４０％となるように脂質相の流速と水相の流速を調節した。具体的には、脂質相の流速および水相の流速はそれぞれ、５０μＬ／分および４５０μＬ／分、１００μＬ／分および４００μＬ／分、１５０μＬ／分および３５０μＬ／分、または２００μＬ／分および３００μＬ／分とした。上記流速比条件それぞれで粒子を調製した（図２）。図２に示されるように、エタノール希釈濃度が低くなるにつれて粒子径が小さくなった。また、図２に示されるように、エタノール希釈濃度が２０％～３０％の時、ＰｄＩは約０．１２と単分散であったが、エタノール希釈濃度１０％ではＰｄＩ　＝　０．３８４±０．０３７７となり、他の希釈濃度よりも多分散性が増した。 (Iv) Flow Velocity Ratio The flow velocity ratio was adjusted so that the ethanol dilution concentration was 10%, 20%, 30%, or 40% in the lipid phase and the aqueous phase. Specifically, the flow rates of the lipid phase and the aqueous phase are 50 μL / min and 450 μL / min, 100 μL / min and 400 μL / min, 150 μL / min and 350 μL / min, or 200 μL / min and 300 μL / min, respectively. did. Particles were prepared under each of the above flow rate ratio conditions (Fig. 2). As shown in FIG. 2, the particle size decreased as the ethanol dilution concentration decreased. Further, as shown in FIG. 2, when the ethanol dilution concentration was 20% to 30%, PdI was monodisperse at about 0.12, but when the ethanol dilution concentration was 10%, PdI = 0.384 ± 0. It was 0377, which was more polydisperse than other dilutions.
　マイクロ流路で調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒ溶液は、２時間の透析を行った（図１）。透析では、Ｓｐｅｃｔｒａｍ　Ｌａｂｐｒａｔｏｒｉｅｓ社の透析クリップＳｐｅｃｔｒａ／Ｐｏｒ　Ｃｌｏｓｕｒｅｓおよび透析膜Ｓｐｅｃｔｒａ／Ｐｏｒ　４　ｄｉａｌｙｓｉｓ　ｍｅｍｂｒａｎｅ　（ＭＷＣＯ　１２ｋ－１４ｋ）を用いた。脂質ナノ粒子溶液は、透析を行うことで透析前と比較して粒子径が減少し、ＰｄＩが増加する傾向にあった（図３）。肝臓を標的としたＤＤＳでは、粒子径が１００　ｎｍ以下の脂質ナノ粒子は目的物質の効率の良い送達を可能とし、粒子径が小さいほどその効率が高まることが示されている。今回検証した条件の中で、特にエタノール希釈濃度２０％の時、粒子径　＝　４７．０±６．０　ｎｍ、ＰｄＩ　＝　０．２４３±０．０１０６の粒子を調製でき、この条件下で得られた粒子を以下の実施例において用いた。このような小さな粒子径を有する分散体の作製も、このような小さなＰｄＩを有する分散体の作製も、従来法では不可能であった。 The CoQ 10- MITO-Porter solution prepared in the microchannel was dialyzed for 2 hours (Fig. 1). For dialysis, dialysis clips Spectra / Por Closures and dialysis membrane Spectra / Por 4 dialysis membrane (MWCO 12k-14k) manufactured by Spectram Laboratories were used. The lipid nanoparticle solution tended to have a smaller particle size and an increased PdI due to dialysis as compared with before dialysis (FIG. 3). In DDS targeting the liver, it has been shown that lipid nanoparticles having a particle size of 100 nm or less enable efficient delivery of the target substance, and that the smaller the particle size, the higher the efficiency. Among the conditions verified this time, especially when the ethanol dilution concentration was 20%, particles having a particle size of 47.0 ± 6.0 nm and PdI = 0.243 ± 0.0106 could be prepared and obtained under these conditions. The particles were used in the following examples. Neither the preparation of a dispersion having such a small particle size nor the preparation of a dispersion having such a small PdI was possible by the conventional method.
１－２．　透析温度の影響
　４℃は生理活性物質が失活せず、衛生面からも無菌的である。一方、２５℃は温度管理が容易で、低コストで扱いが簡便である。そこで透析温度は４℃と２５℃を試みた。その結果、透析温度の違いによる粒子径とＰｄＩの変化は見られず、同様であった（図４）。これは温度に依存症せず、良好な粒子を得ることができるので、調製したい粒子の状況に合わせて温度を選択できることになる。以降の検討は、透析温度は室温とした。 1-2. Effect of dialysis temperature At 4 ° C, physiologically active substances are not inactivated and are sterile from a hygienic point of view. On the other hand, at 25 ° C, temperature control is easy, low cost and easy handling. Therefore, the dialysis temperatures were tried at 4 ° C and 25 ° C. As a result, no change in particle size and PdI was observed due to the difference in dialysis temperature, which was the same (Fig. 4). Since this is temperature-independent and good particles can be obtained, the temperature can be selected according to the situation of the particles to be prepared. In the subsequent studies, the dialysis temperature was room temperature.
１－３．　安定性試験
　調製した粒子の安定性を評価するために、４℃および２５℃、遮光下にて１４日間の安定性試験を行った（図５）。保存期間中、２５℃においては経日的に粒子径が増大した。一方、４℃では粒子径が約５０　ｎｍ付近を維持し、安定であった。 1-3. Stability Test In order to evaluate the stability of the prepared particles, a stability test was conducted at 4 ° C. and 25 ° C. under shading for 14 days (Fig. 5). During the storage period, the particle size increased over time at 25 ° C. On the other hand, at 4 ° C., the particle size was maintained at around 50 nm and was stable.
比較例１
従来法を用いたＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの調製法
　エタノール希釈法（非特許文献２）利用して粒子調製を行った。７．５　ｍＭ　ＤＯＰＥ（ＥｔＯＨ）、７．５　ｍＭ　ＳＭ（ＥｔＯＨ）、７．５　ｍＭ　ＤＭＧ－ＰＥＧ　２０００（ＥｔＯＨ）を調製し、室温とする。エッペンドルフチューブに右図の体積比となるように脂質材料を含有したエタノール懸濁液を準備した。ＣｏＱ１０は懸濁状態にあり沈殿が進むため、使用直前に超音波処理を行った。本懸濁液にＰＢＳ（－）緩衝液を加え、エタノール濃度が９０％となるように希釈した。緩衝液添加後３秒間攪拌をし、その後すぐにエタノール濃度が５　％となるように緩衝液中に懸濁液を添加した。この溶液を限外ろ過フィルターとしてアミコン（ＭＷＣＯ：１００　ｋＤａ）にアプライし、さらに緩衝液を加え限外ろ過を行った（１０００　ｇ、　２５℃、　２０分）。限外ろ過操作は２回繰り返し、エタノール濃度が０．１％になるようにした。回収した脂質ナノ粒子に、脂質量の１０％になるように２　ｍｇ／ｍＬ　ＳＴＲ－Ｒ８を添加・インキュベーション（室温、　３０分）し、Ｒ８－ＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒ（従来法）を調製した。 Comparative Example 1
Preparation method of CoQ 10- MITO-Porter using the conventional method Particles were prepared using the ethanol dilution method (Non-Patent Document 2). Prepare 7.5 mM DOPE (EtOH), 7.5 mM SM (EtOH), and 7.5 mM DMG-PEG 2000 (EtOH) and bring to room temperature. An ethanol suspension containing a lipid material was prepared in an Eppendorf tube so as to have the volume ratio shown on the right. Since CoQ 10 is in a suspended state and precipitation proceeds, ultrasonic treatment was performed immediately before use. PBS (-) buffer was added to the suspension and diluted to an ethanol concentration of 90%. After adding the buffer, the mixture was stirred for 3 seconds, and immediately after that, the suspension was added to the buffer so that the ethanol concentration became 5%. This solution was applied to Amicon (MWCO: 100 kDa) as an ultrafiltration filter, and a buffer solution was further added for ultrafiltration (1000 g, 25 ° C., 20 minutes). The ultrafiltration operation was repeated twice so that the ethanol concentration became 0.1%. To the recovered lipid nanoparticles, 2 mg / mL STR-R8 was added and incubated (room temperature, 30 minutes) so as to have 10% of the lipid amount, and R8-CoQ 10- MITO-Porter (conventional method) was prepared. ..
　粒子径、ＰｄＩ（多分散性指数）およびゼータ電位（表面電位）を測定し、正に帯電する約１００　ｎｍ（ＰｄＩ＝０．３３６±０．００２）のナノ粒子であることを確認した（図７）。調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒ液（従来法）に含まれるＣｏＱ１０濃度とＣｏＱ１０回収率は、ＨＰＬＣを用いて定量し、検量線から算出した。ＨＰＬＣはＡｇｉｌｅｎｔ　１２００　ｓｅｒｉｅｓを使用した。カラムは、ＣＯＳＭＯＳＩＬ（５Ｃ１８ＡＲ－ＩＩ、　４．６×２５０　ｍｍ）を使用し、測定波長：２７５　ｎｍ、　カラム温度：３５℃、　移動相：エタノール／　アセトニトリル＝３／　７、　注入量：５０μＬ、　保持時間＝７　分と測定条件を設定した。その値は、４１４．２±７．１μＭおよび６２．５±４．７　％であった（図６）。また従来法では粒子が調製できず、凝集が生じるため、限外ろ過が困難な例が多くみられた。その原因として、調製時の難水溶性分子ＣｏＱ１０の凝集が挙げられる。マイクロ流路を用いて得られたＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒをネガティブ染色し、透過電子顕微鏡を用いて観察した。結果は図１６に示される通りであった。図１６に示されるように、得られたＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒは、粒子の内部に脂質膜と考えられる構造体を含んでいた。このように、マイクロ流路を用いて得られたＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒは非中空の脂質構造体であることが明らかであった。ＣｏＱ１０が難水溶性であることから、ＣｏＱ１０は脂質膜中に取り込まれたものと考えられ、内部が脂質膜で充填された非中空の脂質構造体は、大量のＣｏＱ１０の内包に適していると考えられた。 The particle size, PdI (multidispersity index) and zeta potential (surface potential) were measured, and it was confirmed that the nanoparticles were positively charged at about 100 nm (PdI = 0.336 ± 0.002) (Fig.). 7). The CoQ 10 concentration and the CoQ 10 recovery rate contained in the prepared CoQ 10- MITO-Porter solution (conventional method) were quantified using HPLC and calculated from the calibration curve. For HPLC, Agilent 1200 series was used. The column uses COSMOSIL (5C18AR-II, 4.6 × 250 mm), measurement wavelength: 275 nm, column temperature: 35 ° C., mobile phase: ethanol / acetonitrile = 3/7, injection volume: 50 μL, retention time. The measurement conditions were set to = 7 minutes. The values were 414.2 ± 7.1 μM and 62.5 ± 4.7% (Fig. 6). In addition, there were many cases in which ultrafiltration was difficult because particles could not be prepared by the conventional method and aggregation occurred. The cause is the aggregation of the poorly water-soluble molecule CoQ 10 at the time of preparation. The CoQ 10- MITO-Porter obtained using the microchannel was negatively stained and observed using a transmission electron microscope. The results were as shown in FIG. As shown in FIG. 16, the obtained CoQ 10- MITO-Porter contained a structure considered to be a lipid membrane inside the particles. As described above, it was clear that the CoQ 10- MITO-Porter obtained by using the microchannel was a non-hollow lipid structure. Since CoQ 10 is poorly water-soluble, it is considered that CoQ 10 was incorporated into the lipid membrane, and the non-hollow lipid structure whose inside is filled with the lipid membrane is suitable for encapsulation of a large amount of CoQ 10. Was thought to be.
実施例２
２－１．　マイクロ流路を用いた粒子調製の精度
　実施例１及び比較例１の結果を比較した。本発明の方法では、マイクロ流路を用いて調製された分散体では、（Ａ）粒子径が小さく、（Ｂ）ＰｄＩも小さい傾向にあった（図７）。特に透析後も、得られた分散体は均一な精度の高い粒子が維持できた。得られた分散体は（Ｃ）ゼータ電位においても約＋２０　ｍＶであり、調製毎のばらつきはなく、ほぼ一定してＳＴＲ－Ｒ８で修飾できたと考えられる。比較例１では、分散体を形成後に分散体をＳＴＲ－Ｒ８で修飾させており、ゼータ電位を約＋２０　ｍＶで再現よく調製することは難しかった。さらに（Ｄ）ＣＶ値は、粒子調製の精度パラメータとして用いた。分散体の粒子径のＣＶは比較例１では０．０８２であったのに対し、本発明の方法の透析前および透析後の分散体の粒子径のＣＶはそれぞれ０．０６２および０．０６５であった。分散体のＰｄＩのＣＶは比較例１では０．１９２であったのに対し、本発明の方法の透析前および透析後の分散体のＰｄＩのＣＶはそれぞれ０．１４８および０．０６６であった。得られた分散体のゼータ電位のＣＶは従来法では０．１７１であったのに対し、本発明の方法透析前および透析後の分散体のゼータ電位のＣＶはそれぞれ０．１１２、０．１１４であった。本発明の方法を用いることにより粒子径は４８．３±３．１　ｎｍと従来技術より１／２倍小さな粒子となった。ＰｄＩも本発明の方法の方が比較例１より小さくなった。マイクロ流路を用いた調製により、ゼータ電位においても＋２１．５±１．８ｍＶとなり、ＳＴＲ－Ｒ８による修飾効果が期待され、細胞への導入や動物への投与に有用であると考えられた。このように、マイクロ流路は、分散体の製造に有用であると共に、分散体を機能性素子で安定に修飾するデバイスとしても有益があると示された。また、粒子径、ＰｄＩおよびゼータ電位のそれぞれのＣＶ値において、本発明の方法で得られる分散体が比較例１で得られた分散体よりも小さかった。これは、本発明の方法によれば、より均一な粒子が調製できていること、すなわち、調製の精度が高いことを意味する。また、マイクロ流路で得られたＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒは、脂質組成から脂質修飾されたポリエチレングリコールを除いて調製した場合には、得られる液体が白濁した。脂質修飾されたポリエチレングリコールは、ＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの溶液への溶解性を向上させることに寄与していると考えられる。 Example 2
2-1. Accuracy of particle preparation using microchannel The results of Example 1 and Comparative Example 1 were compared. In the method of the present invention, in the dispersion prepared using the microchannel, (A) the particle size tended to be small, and (B) PdI also tended to be small (FIG. 7). In particular, even after dialysis, the obtained dispersion was able to maintain uniform and highly accurate particles. The obtained dispersion was about +20 mV even at the (C) zeta potential, and it is considered that there was no variation between preparations and that the dispersion could be modified with STR-R8 almost constantly. In Comparative Example 1, the dispersion was modified with STR-R8 after the dispersion was formed, and it was difficult to reproducibly prepare the zeta potential at about +20 mV. Furthermore, the (D) CV value was used as an accuracy parameter for particle preparation. The particle size CV of the dispersion was 0.082 in Comparative Example 1, whereas the particle size CV of the dispersion before and after dialysis of the method of the present invention was 0.062 and 0.065, respectively. there were. The CV of the PdI of the dispersion was 0.192 in Comparative Example 1, whereas the CV of the PdI of the dispersion before and after dialysis of the method of the present invention was 0.148 and 0.066, respectively. .. The CV of the zeta potential of the obtained dispersion was 0.171 in the conventional method, whereas the CV of the zeta potential of the dispersion before and after dialysis by the method of the present invention was 0.112 and 0.114, respectively. Met. By using the method of the present invention, the particle size is 48.3 ± 3.1 nm, which is 1/2 times smaller than that of the prior art. The PdI of the method of the present invention was also smaller than that of Comparative Example 1. By the preparation using the microchannel, the zeta potential was +21.5 ± 1.8 mV, and the modifying effect by STR-R8 was expected, which was considered to be useful for introduction into cells and administration to animals. As described above, it has been shown that the microchannel is useful for producing a dispersion and also as a device for stably modifying the dispersion with a functional element. In addition, the dispersion obtained by the method of the present invention was smaller than the dispersion obtained in Comparative Example 1 at each CV value of particle size, PdI and zeta potential. This means that according to the method of the present invention, more uniform particles can be prepared, that is, the accuracy of preparation is high. Further, when CoQ 10- MITO-Porter obtained in the microchannel was prepared by removing lipid-modified polyethylene glycol from the lipid composition, the obtained liquid became cloudy. Lipid-modified polyethylene glycol is considered to contribute to improving the solubility of CoQ 10- MITO-Porter in the solution.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　さらに表１に示すように、調製量も比較例１（従来技術）ではμＬオーダーであったが、本発明の方法では無制限に調製が可能で実験室レベルでもＬオーダーという大量調製が見込める。調製時間も大容量になるほど本発明の方法の方が、短い時間で調製ができる。したがって、マイクロ流路を用いた本発明の方法での調製は、小さな粒径を有する難溶性化合物を含む脂質膜構造体の分散体を調製できた。また当該分散体は、小さなＰｄＩを有した。さらには脂質ナノ粒子形成時間が大幅に短縮された。したがって、製剤の製造時間およびコストが低減されると考えられる。また、材料を連続的にマイクロ流路に供給することによって、連続的な脂質膜構造体の分散体を得ることができた。 Furthermore, as shown in Table 1, the amount of preparation was also on the order of μL in Comparative Example 1 (conventional technique), but the method of the present invention allows unlimited preparation, and a large amount of preparation on the order of L can be expected even at the laboratory level. The method of the present invention can be prepared in a shorter time as the preparation time becomes larger. Therefore, the preparation by the method of the present invention using the microchannel could prepare a dispersion of a lipid membrane structure containing a sparingly soluble compound having a small particle size. The dispersion also had a small PdI. Furthermore, the lipid nanoparticle formation time was significantly reduced. Therefore, it is considered that the production time and cost of the preparation are reduced. In addition, by continuously supplying the material to the microchannel, a continuous dispersion of lipid membrane structures could be obtained.
実施例３
３－１．　細胞内取り込み評価（１）
　実施例１で調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの細胞内導入能を評価するために、蛍光標識（ニトロベンゾオキサジアゾール（ＮＢＤ）－ＤＯＰＥを脂質量の０．５　ｍｏｌ％修飾）を施したキャリアを調製し、子宮頸がんＨｅＬａ細胞への取込みをフローサイトメトリー（ＦＡＣＳ）を用いて評価し、共焦点レーザー顕微鏡（ＣＬＭＳ）による細胞内局在観察を行った。フローサイトメトリーを用いた細胞取り込み評価により、本発明の方法で得られた分散体は従来法と比較して細胞内への取り込みが多いことが示された（図８）。また、共焦点レーザー顕微鏡によってＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの細胞内動態を観察した結果、従来法と比較して本発明の方法で得られた分散体では、細胞内に多くのシグナルが観察され、細胞への導入効率が優れていることが示された（図９）。さらに、ミトコンドリアへの集積も確認することができた。本発明の方法によって調製した粒子はどの細胞にも従来法よりも多くが到達していた。一方、従来法では、粒子が細胞へ到達しているものとしていないものが観察された。実施例１で調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの分散体の細胞内導入効率およびミトコンドリアへの集積効率の高さは、分散体の粒径が小さく、かつ均質であることに起因していると考えられる。また、従来法で得られる分散体で、細胞に到達しない粒子が生じるのは、分散体の粒径が不均質であり、粒径の大きなものが含まれることに起因していると考えられた。 Example 3
3-1. Evaluation of intracellular uptake (1)
In order to evaluate the intracellular introduction ability of CoQ 10- MITO-Porter prepared in Example 1, fluorescent labeling (nitrobenzoxadiazole (NBD) -DOPE modified with 0.5 mol% of lipid amount) was applied. Carriers were prepared, uptake into cervical cancer HeLa cells was evaluated using flow cytometry (FACS), and intracellular localization was observed with a confocal laser scanning microscope (CLMS). Evaluation of cell uptake using flow cytometry showed that the dispersion obtained by the method of the present invention had a large amount of uptake into cells as compared with the conventional method (Fig. 8). In addition, as a result of observing the intracellular dynamics of CoQ 10- MITO-Porter with a confocal laser scanning microscope, many signals were observed inside the cells in the dispersion obtained by the method of the present invention as compared with the conventional method. It was shown that the efficiency of introduction into cells was excellent (Fig. 9). Furthermore, accumulation in mitochondria could be confirmed. More particles prepared by the method of the present invention reached any cell than the conventional method. On the other hand, in the conventional method, some particles were observed to reach the cells and some did not. The high efficiency of intracellular introduction and accumulation of the CoQ 10- MITO-Porter dispersion prepared in Example 1 into mitochondria is attributed to the small particle size and homogeneity of the dispersion. Conceivable. In addition, it is considered that the reason why particles that do not reach the cells are generated in the dispersion obtained by the conventional method is that the particle size of the dispersion is inhomogeneous and contains a large particle size. ..
３－３．　細胞内取り込み評価（３）
　細胞としてミトコンドリア病態モデル細胞を用いたこと以外は、細胞内取り込み評価（１）と同様の方法により、共焦点レーザー顕微鏡（ＣＬＭＳ）による細胞内局在観察を行った。この場合にも同様の傾向が観察された（図１０）。図１０に示されるように、ミトコンドリア病を呈する細胞に対しても、実施例１で調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの分散体は、良好に細胞内に導入され、ミトコンドリアに蓄積した。 3-3. Evaluation of intracellular uptake (3)
Intracellular localization was observed with a confocal laser scanning microscope (CLMS) by the same method as in intracellular uptake evaluation (1) except that mitochondrial pathological model cells were used as cells. A similar tendency was observed in this case as well (Fig. 10). As shown in FIG. 10, the dispersion of CoQ 10- MITO-Porter prepared in Example 1 was satisfactorily introduced into the cells and accumulated in the mitochondria even in the cells exhibiting mitochondrial disease.
３－３．　細胞内取り込み評価（３）
　細胞としてヒト心筋由来細胞（ｈｕｍａｎ　ＣＤＣ）を用いたこと以外は、細胞内取り込み評価（１）と同様の方法により、共焦点レーザー顕微鏡（ＣＬＭＳ）による細胞内局在観察を行った。ｈｕｍａｎ　ＣＤＣは先天性心疾患の根治手術の際に切り取られた余剰心室筋から単離した。結果を図１１に示す。図１１に示されるように、先天性心疾患患者のＣＤＣに対しても、実施例１で調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの分散体は、良好に細胞内に導入され、ミトコンドリアに蓄積した。 3-3. Evaluation of intracellular uptake (3)
Intracellular localization was observed with a confocal laser scanning microscope (CLMS) by the same method as in the intracellular uptake evaluation (1) except that human myocardial cells (human CDC) were used as cells. The human CDC was isolated from the surplus ventricular muscle that was excised during radical surgery for congenital heart disease. The results are shown in FIG. As shown in FIG. 11, even for congenital heart disease patients CDC, dispersion of CoQ 10 -MITO-Porter prepared in Example 1 is well be introduced intracellularly and accumulated in the mitochondria.
３－４．　細胞内取り込み評価（４）
　細胞としてヒト肺動脈平滑筋細胞（Ｈｕｍａｎ　ｐｕｌｍｏｎａｒｙ　ａｒｔｅｒｙ　ｓｍｏｏｔｈ　ｍｕｓｃｌｅ　ｃｅｌｌｓ）を用いたこと以外は、細胞内取り込み評価（１）と同様の方法により、共焦点レーザー顕微鏡（ＣＬＭＳ）による細胞内局在観察を行った。細胞内局在観察結果を図１２に示す。図１２に示されるように、１に示す。図１１に示されるように、先天性心疾患患者のＣＤＣに対しても、実施例１で調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの分散体は、良好に細胞内に導入され、ミトコンドリアに蓄積した。 3-4. Evaluation of intracellular uptake (4)
Intracellular localization observation was performed by a confocal laser scanning microscope (CLMS) by the same method as in the intracellular uptake evaluation (1) except that human pulmonary artery smooth muscle cells (Human pulmonary artery smooth muscle cells) were used as cells. It was. The results of intracellular localization observation are shown in FIG. As shown in FIG. 12, it is shown in 1. As shown in FIG. 11, even for congenital heart disease patients CDC, dispersion of CoQ 10 -MITO-Porter prepared in Example 1 is well be introduced intracellularly and accumulated in the mitochondria.
実施例４
ＣｏＱ１０濃度の変化
　実施例１で採用したＣｏＱ１０の初期量（１．５　ｍＭ　２００μＬ、　以下、１　ＣｏＱ１０）が脂質に対して妥当な濃度であるのか確かめるために、ＣｏＱ１０濃度を１／２倍（０．７５　ｍＭ、　以下、１／２　ＣｏＱ１０）、２倍（３　ｍＭ、　以下、２　ＣｏＱ１０）にして検証した。マイクロ流路でのエタノール希釈濃度は２０％とした。透析前の粒子径は実施例１の濃度（１　ＣｏＱ１０）が最も小さく、７０．５±０．３　ｎｍであり、ＰｄＩも最も小さかった（図１３）。透析後はどのＣｏＱ１０濃度も粒子径は、約５０　ｎｍであり、ＰｄＩも約０．２となり、透析前よりも増加する傾向にあった。ＣｏＱ１０濃度は、それぞれ１／２　ＣｏＱ１０透析前が５８．１±５．０μＭ、１／２　ＣｏＱ１０透析後が３６．０±１．５μＭ、１　ＣｏＱ１０透析前が１１３．３±１２．７μＭ、１　ＣｏＱ１０透析後が７６．１±７．５μＭ、２　ＣｏＱ１０透析前が２６１．８±１４．８μＭ、２　ＣｏＱ１０透析後が１７６．４±１０．８μＭであった。回収率は、それぞれ１／２　ＣｏＱ１０透析前が８３．３±７．１％、１／２　ＣｏＱ１０透析後が６７．２±３．４％、１　ＣｏＱ１０透析前が８１．２±９．１％、１　ＣｏＱ１０透析後が７２．８±６．７％、２　ＣｏＱ１０透析前が９３．８±５．３％、２　ＣｏＱ１０透析後が８０．５±４．７％であった。そしてＤｒｕｇ／Ｌｉｐｉｄ（ｗ／ｗ）は、それぞれ１／２　ＣｏＱ１０透析前が０．１０±０．０１、１／２　ＣｏＱ１０透析後が０．０９±０．００、１　ＣｏＱ１０透析前が０．２０±０．０３、１　ＣｏＱ１０透析後が０．１９±０．０３、２　ＣｏＱ１０透析前が０．４８±０．０６、２　ＣｏＱ１０透析後が０．４６±０．０６であった。したがって、透析をすることで回収率やＤｒｕｇ／Ｌｉｐｉｄは減少するが、仕込むＣｏＱ１０濃度を増加させると回収率やＤｒｕｇ／Ｌｉｐｉｄも共に増加する傾向にあった。 Example 4
Change in CoQ 10 concentration In order to confirm whether the initial amount of CoQ 10 (1.5 mM 200 μL, hereinafter, 1 CoQ 10) adopted in Example 1 is an appropriate concentration for lipids, the CoQ 10 concentration is halved. It was verified by doubling (0.75 mM, hereinafter, 1/2 CoQ10) and doubling (3 mM, hereinafter, 2 CoQ10). The ethanol dilution concentration in the microchannel was 20%. The particle size before dialysis was the smallest in the concentration of Example 1 (1 CoQ 10 ), 70.5 ± 0.3 nm, and the PdI was also the smallest (FIG. 13). After dialysis, the particle size of all CoQ 10 concentrations was about 50 nm, and the PdI was about 0.2, which tended to increase from that before dialysis. The CoQ 10 concentration was 58.1 ± 5.0 μM before 1/2 CoQ10 dialysis, 36.0 ± 1.5 μM after 1/2 CoQ10 dialysis, and 113.3 ± 12.7 μM before CoQ10 dialysis, respectively. It was 76.1 ± 7.5 μM after CoQ10 dialysis, 261.8 ± 14.8 μM before CoQ10 dialysis, and 176.4 ± 10.8 μM after CoQ10 dialysis. The recovery rates were 83.3 ± 7.1% before 1/2 CoQ10 dialysis, 67.2 ± 3.4% after 1/2 CoQ10 dialysis, and 81.2 ± 9.1% before 1 CoQ10 dialysis, respectively. 1, 72.8 ± 6.7% after CoQ10 dialysis, 93.8 ± 5.3% before 2 CoQ10 dialysis, and 80.5 ± 4.7% after 2 CoQ10 dialysis. The Drag / Lipid (w / w) is 0.10 ± 0.01 before 1/2 CoQ10 dialysis, 0.09 ± 0.00 after 1/2 CoQ10 dialysis, and 0.20 before 1 CoQ10 dialysis, respectively. It was ± 0.03, 0.19 ± 0.03 after 1 CoQ10 dialysis, 0.48 ± 0.06 before 2 CoQ10 dialysis, and 0.46 ± 0.06 after 2 CoQ10 dialysis. Therefore, the recovery rate and the drug / lipid decreased by dialysis, but the recovery rate and the drug / lipid tended to increase as the concentration of CoQ 10 to be charged was increased.
実施例５
調製量による比較
　従来法（比較例１）と本発明の方法（マイクロ流路を用いた調製法）において調製量による物性の変化が見られるか検証した。従来法では、１回の調製量に依存して粒子径、ＰｄＩ、およびゼータ電位（ＺＰ）が大きく変動した。本発明の方法では粒子径およびＰｄＩ共に従来法に比べ、調製量の影響を受けにくく、安定した調製が可能であった（表２）。
　また従来法では、安定したＳＴＲ－Ｒ８の修飾を行うためには１００μＬという少量単位でなければならない。しかし、調製容量を増大させ４００μＬの容量でＳＴＲ－Ｒ８を修飾しようとすると、ゼータ電位が９．８±０．９　ｍＶとなり、小容量の調製時よりも電位が下がってしまう。これは、脂質ナノ粒子医薬品をスケールアップする際の大きな障壁となる。一方、マイクロ流路を用いた調製法では、調製量に依存せず、ゼータ電位が約２０　ｍＶという良好な値を示し、ＣＶ値（ばらつきの指標）も透析前は０．０７２、透析後は０．０３１と従来法よりも小さい。以上より、マイクロ流路を用いた調製法は、ＳＴＲ－Ｒ８などの機能性素子を安定に修飾でき、脂質ナノ粒子医薬品の製造には不可欠であることが示された。 Example 5
Comparison by Preparation Amount It was verified whether the physical properties changed depending on the preparation amount between the conventional method (Comparative Example 1) and the method of the present invention (preparation method using a microchannel). In the conventional method, the particle size, PdI, and zeta potential (ZP) fluctuate greatly depending on the amount prepared at one time. In the method of the present invention, both the particle size and PdI were less affected by the amount of preparation as compared with the conventional method, and stable preparation was possible (Table 2).
Further, in the conventional method, in order to perform stable modification of STR-R8, the unit must be as small as 100 μL. However, if the preparation volume is increased and an attempt is made to modify STR-R8 with a volume of 400 μL, the zeta potential becomes 9.8 ± 0.9 mV, which is lower than that at the time of preparation of a small volume. This is a major barrier to scaling up lipid nanoparticle drugs. On the other hand, in the preparation method using the microchannel, the zeta potential shows a good value of about 20 mV regardless of the prepared amount, and the CV value (index of variation) is 0.072 before dialysis and 0.072 after dialysis. It is 0.031, which is smaller than the conventional method. From the above, it was shown that the preparation method using the microchannel can stably modify functional elements such as STR-R8 and is indispensable for the production of lipid nanoparticle pharmaceuticals.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
実施例６
６－１．　ＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの濃縮
　実施例１で得られた分散体の濃縮を試みた。従来法では、ＣｏＱ１０濃度を５０６．９±１３４．１μＭまで濃縮できるものの、限外ろ過フィルターに目詰まりが生じやすく、効率的な濃縮操作が行えない場合があった。そこで、本実施例では、１つの脂質ナノ粒子あたりの薬力価を高めるために、実施例１で得られた分散体を濃縮することで、高濃度ＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの調製を試みた。実施例１で得られた分散体の透析後の溶液をアミコン（ＭＷＣＯ：１００　ｋＤａ）にアプライし、限外ろ過を行った（１０００　ｇ、　２５℃、　２５分）。その結果、溶液量が１、５００μＬから１、０００μＬまでしか濃縮されず、良好な濃縮操作が行えないと思われた（通常は４００μＬまで濃縮される）。したがって、透析後の溶液を遠心しただけでは効率的な濃縮は見込めなかった。その原因として、ＣｏＱ１０の分散性が低下し、限外ろ過フィルターに目詰まりが生じた可能性がある。そこでＣｏＱ１０の分散性を向上させるために、ＰＢＳ（－）添加による希釈を行うことで限外ろ過による濃縮が可能か検証した（図１４）。 Example 6
6-1. Concentration of CoQ 10- MITO-Porter An attempt was made to concentrate the dispersion obtained in Example 1. In the conventional method, the CoQ 10 concentration can be concentrated to 506.9 ± 134.1 μM, but the ultrafiltration filter is likely to be clogged, and an efficient concentration operation may not be possible. Therefore, in this example, in order to increase the drug titer per lipid nanoparticles, an attempt was made to prepare a high-concentration CoQ 10- MITO-Porter by concentrating the dispersion obtained in Example 1. .. The post-dialysis solution of the dispersion obtained in Example 1 was applied to Amicon (MWCO: 100 kDa) and ultrafiltered (1000 g, 25 ° C., 25 minutes). As a result, the amount of the solution was concentrated only from 1,500 μL to 1,000 μL, and it seemed that a good concentration operation could not be performed (usually, it was concentrated to 400 μL). Therefore, efficient concentration could not be expected only by centrifuging the solution after dialysis. The cause may be that the dispersibility of CoQ 10 is lowered and the ultrafiltration filter is clogged. Therefore, in order to improve the dispersibility of CoQ 10 , it was verified whether it is possible to concentrate by ultrafiltration by diluting by adding PBS (-) (Fig. 14).
６－２．　ＰＢＳ（－）添加
　透析後の溶液を限外ろ過フィルターに１５００μＬアプライし、４℃または２５℃のＰＢＳ（－）を３．５　ｍＬ（全量５　ｍＬ）、８．５　ｍＬ（全量１０　ｍＬ）、および１３．５　ｍＬ（全量１５　ｍＬ）添加させ、分散性を向上させてから遠心操作を行った。４℃のＰＢＳ（－）を用いると、添加量が増大するとＰｄＩも増大した。一方、２５℃のＰＢＳ（－）を添加させた時はＰＢＳを添加されていない時と物性に大きな変化は見られなかった。２５℃のＰＢＳ（－）８．５　ｍＬを添加させた際のＣｏＱ１０濃度は４８７．０μＭ、ＣｏＱ１０回収率は２９．２％であった（図１４）。このようにして、高濃度ＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒを調製した。本発明の方法で得られた分散体は、粒径が均質であり、限外ろ過フィルターの目詰まりによる、ろ過効率の悪化は認められなかった。また、これによって、低いＣｏＱ１０濃度の溶液を用いてＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒを調製し、その後、濃縮するＣｏＱ１０製剤の調製戦略の有効性が明らかとなった。 6-2. Addition of PBS (-) Apply 1500 μL of the solution after dialysis to an ultrafiltration filter, and add 3.5 mL (total volume 5 mL), 8.5 mL (total volume 10 mL) of PBS (-) at 4 ° C or 25 ° C. And 13.5 mL (15 mL in total) was added to improve dispersibility, and then centrifugation was performed. When PBS (−) at 4 ° C. was used, PdI also increased as the amount added increased. On the other hand, when PBS (-) at 25 ° C. was added, there was no significant change in physical properties from when PBS (-) was not added. When 8.5 mL of PBS (−) at 25 ° C. was added, the CoQ 10 concentration was 487.0 μM, and the CoQ 10 recovery rate was 29.2% (Fig. 14). In this way, a high concentration CoQ 10- MITO-Porter was prepared. The dispersion obtained by the method of the present invention had a uniform particle size, and no deterioration in filtration efficiency was observed due to clogging of the ultrafiltration filter. This also revealed the effectiveness of the preparation strategy for CoQ 10 formulations in which a low CoQ 10 concentration solution was used to prepare CoQ 10- MITO-Porter and then concentrated.
６－３．　安定性試験
　調製した粒子の安定性を評価するために、４℃、遮光下にて１４日間の安定性試験を行った（図１５）。保存期間中、粒子径は約５０　ｎｍ付近を維持し、濃縮操作を行っても安定なＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒであった。 6-3. Stability test In order to evaluate the stability of the prepared particles, a stability test was conducted at 4 ° C. under shading for 14 days (Fig. 15). During the storage period, the particle size was maintained at about 50 nm, and CoQ 10- MITO-Porter was stable even after the concentration operation.
実施例７
実施例７－１．　本発明のＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒによるミトコンドリア呼吸能の改善効果
　マイクロ流路デバイスにて調製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒによる治療効果を検証するために、ミトコンドリア機能不全であるＭＥＬＡＳの線維芽細胞、Ｌｅｉｇｈの線維芽細胞、ＬＨＯＮの線維芽細胞、および正常な線維芽細胞を用いてミトコンドリア呼吸能評価を行った。細胞数は、ＭＥＬＡＳの線維芽細胞は２．５×１０４細胞／ウェルとし、それ以外の細胞は２×１０４細胞／ウェルとした。測定は、常法により行った。具体的には、測定の２４±３時間前にＡｇｉｌｅｎｔ　Ｓｅａｈｏｒｓｅ　ＸＦｐ　Ｃｅｌｌ　Ｃｕｌｔｕｒｅ　Ｍｉｎｉｐｌａｔｅｓ　（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ、　Ｓａｎｔａ　Ｃｌａｒａ、　ＣＡ、　ＵＳＡ）に播種した　（１００００　ｃｅｌｌｓ／ｗｅｌｌ　程度）。ＸＦ　ＤＭＥＭ　Ｍｅｄｉｕｍ　（Ａｇｉｌｅｎｔ）にあらかじめ　１．０　Ｍ　Ｇｌｕｃｏｓｅ　Ｓｏｌｕｔｉｏｎおよび２００　ｍＭ　Ｇｌｕｔａｍｉｎｅ　Ｓｏｌｕｔｉｏｎ　（Ａｇｉｌｅｎｔ）　を加えて終濃度２４．７５　ｍＭ　グルコース、　４　ｍＭ　グルタミンを含むランニング培地を調製した。測定１時間前から本培地を用いて細胞を培養し（ＣＯ２　ｆｒｅｅ、　３７℃）、Ａｇｉｌｅｎｔ　Ｓｅａｈｏｒｓｅ　ＸＦｐ　ｅｘｔｒａｃｅｌｌｕｌａｒ　ｆｌｕｘ　ａｎａｌｙｚｅｒ　（Ａｇｉｌｅｎｔ　Ｔｅｃｈｎｏｌｏｇｉｅｓ、　Ｓａｎｔａ　Ｃｌａｒａ、　ＣＡ、　ＵＳＡ）　を用いて測定した。試料（ＮＴ（未処理）：　ＰＢＳ（－）、　ＣｏＱ１０　ｓｕｓ（カプセル化していない遊離ＣｏＱ１０）：　７５　μＭ　ＣｏＱ１０　（ＰＢＳ　（－）中　：　ＥｔＯＨ　＝　７　：　３）およびＣｏＱ１０－　ＭＩＴＯ－Ｐｏｒｔｅｒ　（ＣｏＱ１０濃度　：　約　７．５　μＭ））は測定開始後に添加し、３時間インキュベートした（ＣＯ２　フリー、　３７℃）。細胞の酸素消費量（ＯＣＲ）とミトコンドリア機能を評価するためにＸＦｐ　Ｃｅｌｌ　Ｍｉｔｏ　Ｓｔｒｅｓｓ　Ｔｅｓｔ　Ｋｉｔ　（Ａｇｉｌｅｎｔ）を用いて、１　μＭ　オリゴマイシン（電子伝達系　呼吸鎖複合体ＶのＡＴＰ合成酵素阻害剤）を添加し、酸素消費率をベースラインにし、１５分後に１　μＭ　ＦＣＣＰ　（脱共役剤）＋３ｍＭピルビン酸を添加し、最大呼吸能を測定し、さらに１５分後に０．５μＭロテノンおよび０．５μＭアンチマイシンＡ（電子伝達系呼吸鎖複合体ＩおよびＩＩＩの阻害剤）を添加して、再びベースラインの酸素消費量を測定した。最大呼吸能は、酸素消費率（％）の最大値からロテノンおよびアンチマイシンＡ添加後の酸素消費率（％）を控除して求めた。経時的な酸素消費率（％）を図１７Ａに示し、最大呼吸能を図１７Ｂに示した。 Example 7
Example 7-1. Effect of improving mitochondrial respiration by CoQ 10- MITO-Porter of the present invention In order to verify the therapeutic effect of CoQ 10- MITO-Porter prepared by a microchannel device, MELAS fibroblasts with mitochondrial dysfunction, Mitochondrial respiration was evaluated using Leight fibroblasts, LHON fibroblasts, and normal fibroblasts. The number of cells was 2.5 × 10 4 cells / well for MELAS fibroblasts and 2 × 10 4 cells / well for other cells. The measurement was carried out by a conventional method. Specifically, the seeds were sown on Agilent Technologies XFp Cell Culture Minilates (Agilent Technologies, Santa Clara, CA, USA) 24 ± 3 hours before the measurement (about 10000 cells / well). A running medium containing a final concentration of 24.75 mM glucose and 4 mM glutamine was prepared by adding 1.0 M Glucose Solution and 200 mM Glutamine Solution (Agilent) in advance to XF DMEM Medium (Agilent). Cells were cultured using this medium (CO 2 free, 37 ° C.) from 1 hour before the measurement, and measured using Agilent Technologies XFp extracellular flux analysers (Agilent Technologies, Santa Clara, CA, USA). Samples (NT (untreated): PBS (-), CoQ 10 sus (unencapsulated free CoQ 10 ): 75 μM CoQ 10 (in PBS (-): EtOH = 7: 3) and CoQ 10 -MITO-Porter (CoQ 10 concentration: about 7.5 μM)) was added after the start of measurement and incubated for 3 hours (CO 2 free, 37 ° C.). 1 μM oligomycin (an ATP synthase inhibitor of electron transport chain respiratory chain complex V) was used to evaluate cellular oxygen consumption (OCR) and mitochondrial function using the XFp Cell Mito Stress Test Kit (Agient). Add, base oxygen consumption, add 1 μM FCCP (deconjugating agent) + 3 mM pyruvate after 15 minutes, measure maximal respiration, and after 15 minutes 0.5 μM rotenone and 0.5 μM antimycin. A (an inhibitor of electron transport chain respiratory chain complexes I and III) was added and baseline oxygen consumption was measured again. The maximum respiratory capacity was determined by subtracting the oxygen consumption rate (%) after the addition of rotenone and antimycin A from the maximum value of the oxygen consumption rate (%). The oxygen consumption rate (%) over time is shown in FIG. 17A, and the maximum respiratory capacity is shown in FIG. 17B.
　結果は図１７ＡおよびＢに示される通りであった。図１７ＡおよびＢに示されるように、カプセル化していないＣｏＱ１０のＰＢＳ（－）懸濁液の添加では、正常細胞では酸素消費率も最大呼吸能も向上が認められなかった。ＭＥＬＡＳの線維芽細胞、Ｌｅｉｇｈの線維芽細胞、およびＬＨＯＮの線維芽細胞では、ＣｏＱ１０のＰＢＳ（－）懸濁液の添加で少しの酸素消費率及び最大呼吸能の向上が認められたが、マイクロ流路を用いて作製したＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒは、それぞれの細胞種の酸素消費率及び最大呼吸能を大きく向上させた。 The results were as shown in FIGS. 17A and 17B. As shown in FIGS. 17A and B, PBS of CoQ 10 unencapsulated (-) in the addition of the suspension, oxygen consumption rate also improves the maximum breathing capacity is not observed in normal cells. In MELAS fibroblasts, Light fibroblasts, and LHON fibroblasts, the addition of a PBS (-) suspension of CoQ 10 showed a slight improvement in oxygen consumption and maximal respiration. CoQ 10- MITO-Porter produced using the microchannel greatly improved the oxygen consumption rate and maximum respiration capacity of each cell type.
７－２．肝障害モデルマウスに対するＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒの治療効果。
　本実施例では、肝障害モデルマウスに対して、マイクロ流路で得られた本発明のＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒを投与してその治療効果を調べた。 7-2. Therapeutic effect of CoQ 10- MITO-Porter on liver disorder model mice.
In this embodiment, for hepatopathy model mice were examined its therapeutic effect by administration of CoQ 10 -MITO-Porter of the present invention obtained in microchannel.
　アセトアミノフェンは、６０℃の加熱条件下でＰＢＳ（－）に溶解させた。図１８Ａに示されるスキームで、マウス（三協ラボサービス社製のＣ５７ＢＬ６、１２～１３週齢、雄、体重約２８ｇ、ｎ＝３）に対して、非絶食条件下で、４００ｍｇ／ｋｇ体重の用量でアセトアミノフェン（富士フィルム和光純薬株式会社）を含むＰＢＳ（－）を腹腔内投与した。１時間後に、マイクロ流路で得られた本発明のＣｏＱ１０－ＭＩＴＯ－ＰｏｒｔｅｒまたはＰＢＳ（－）（陰性対照）をそれぞれ８μＬ／ｇの用量で投与し、２４時間後に心採血および肝臓摘出を行った。 Acetaminophen was dissolved in PBS (−) under heating conditions of 60 ° C. In the scheme shown in FIG. 18A, a mouse (C57BL6 manufactured by Sankyo Lab Service, 12-13 weeks old, male, body weight about 28 g, n = 3) was subjected to 400 mg / kg body weight under non-fasting conditions. PBS (-) containing acetaminophen (Fuji Film Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered at a dose. After 1 hour, CoQ 10- MITO-Porter or PBS (-) (negative control) of the present invention obtained in the microchannel was administered at a dose of 8 μL / g, respectively, and after 24 hours, cardiac blood sampling and liver removal were performed. It was.
　心採血した血液を室温にて１～２時間放置し、遠心した（４℃、　１５分、　３５００　ｒｐｍ）。その後、血清を回収し、トランスアミナーゼ　ＣＩＩ－テストワコー（富士フィルム和光純薬株式会社、　Ｏｓａｋａ、　Ｊａｐａｎ）を用いてＡＬＴを測定し、肝障害の程度を評価した。結果は、図１８Ｂの左パネルに示される通りであった。図１８Ｂの左パネルに示されるように、陰性対照では肝障害のマーカーであるＡＬＴの量が１０００ＩＵ／Ｌ弱であったのに対して、マイクロ流路で得られた本発明のＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒを投与した群では、ＡＬＴ値が大きく低下した。 The collected blood was left at room temperature for 1 to 2 hours and centrifuged (4 ° C., 15 minutes, 3500 rpm). Then, serum was collected and ALT was measured using transaminase CII-Test Wako (Fuji Film Wako Pure Chemical Industries, Ltd., Osaka, Japan) to evaluate the degree of liver damage. The results were as shown in the left panel of FIG. 18B. As shown in the left panel of FIG. 18B, the amount of ALT, which is a marker of liver damage, was less than 1000 IU / L in the negative control, whereas the CoQ 10- MITO of the present invention obtained by the microchannel was obtained. -The ALT value was significantly reduced in the Porter-administered group.
　摘出した肝臓は、組織学的評価に供した。肝臓は４％パラホルムアルデヒド（富士フィルム和光純薬株式会社）にて血抜きをし、４℃で一晩静置した。その後、４時間ごとに４℃で１０％スクロース、２０％スクロース、３０％スクロースを含む溶液で順番に処理し、その後、３０％スクロースを含む溶液で肝臓を一晩静置した。肝臓組織を埋没皿に入れ、組織切片埋没剤Ｏ．Ｃ．Ｔ．Ｃｏｍｐｏｕｎｄ　（サクラファインテックジャパン株式会社）で充填した。さらに液体窒素を用いて組織を凍結させた。凍結した試料は、ＬＥＩＣＡ　ＣＭ３０５０Ｓ　（Ｌｅｉｃａ　Ｂｉｏｓｙｓｔｅｍｓ）にて２０μｍの厚さでスライスし、凍結切片を作製した。
　切り出した組織切片をよく乾燥させ、ヘマトキシリン－エオジン染色（ＨＥ染色）に供した。まず、組織切片をヘマトキシリンにて８分間処理して染色し、１０分間の流水水洗に供した。次に組織切片をエオジンにて３分間処理して染色し、軽く水洗した。その後、組織切片を７０％エタノール、９０％エタノール、１００％エタノール溶液で順番に処理し、脱水した。さらに組織切片をキシロールに浸した。乾燥後、組織切片をソフトマウントを用いて封入した。組織切片の観察は実体顕微鏡ＴＹＰＥ１０１Ｍ（ＳＨＩＭＡＤＺＵ）を用いて行った。
　結果は、図１８Ｂの右パネルに示される組織像の通りであった。 The removed liver was subjected to histological evaluation. The liver was drained with 4% paraformaldehyde (Fuji Film Wako Pure Chemical Industries, Ltd.) and allowed to stand at 4 ° C. overnight. Then, every 4 hours, the liver was treated with a solution containing 10% sucrose, 20% sucrose, and 30% sucrose in order at 4 ° C., and then the liver was allowed to stand overnight with a solution containing 30% sucrose. The liver tissue was placed in an implantation dish, and the tissue section implanter O. C. T. It was filled with Compound (Sakura Finetech Japan Co., Ltd.). In addition, liquid nitrogen was used to freeze the tissue. Frozen samples were sliced with LEICA CM3050S (Leica Biosystems) to a thickness of 20 μm to prepare frozen sections.
The excised tissue sections were thoroughly dried and subjected to hematoxylin-eosin staining (HE staining). First, the tissue sections were treated with hematoxylin for 8 minutes, stained, and subjected to running water for 10 minutes. The tissue sections were then treated with eosin for 3 minutes, stained and lightly washed with water. Then, the tissue section was treated with 70% ethanol, 90% ethanol, and 100% ethanol solution in this order, and dehydrated. Further, the tissue section was immersed in xylene. After drying, tissue sections were encapsulated using a soft mount. Observation of the tissue section was performed using a stereomicroscope TYPE101M (SHIMADZU).
The results were as shown in the right panel of FIG. 18B.
　図１８Ｂの右パネルに示されるように、ＰＢＳ（－）で処置した陰性対照群では、肝臓組織に空間が認められ、組織が破壊されているようすが認められた。これに対して、本発明のＣｏＱ１０－ＭＩＴＯ－Ｐｏｒｔｅｒを投与した群では、組織の破壊が抑制され、正常な肝臓組織像を示した。 As shown in the right panel of FIG. 18B, in the negative control group treated with PBS (-), space was observed in the liver tissue, and it was observed that the tissue was destroyed. On the other hand, in the group to which CoQ 10- MITO-Porter of the present invention was administered, tissue destruction was suppressed and a normal liver histology was shown.
　このように上記実施例では、難水溶性化合物を含有する非中空の脂質構造体を調製することができた。難水溶性化合物としてＣｏＱ１０を用いたところ、ＣｏＱ１０は、細胞内に導入され、細胞の呼吸能を向上させることができた。肝障害モデルマウスにおいて肝障害を治療的に処置することができた。このように、得られた脂質構造体には、細胞膜透過性を付与することができ、また、ミトコンドリア指向性を付与することができ、かつ、導入されたＣｏＱ１０は機能的に有効であった。 Thus, in the above example, a non-hollow lipid structure containing a poorly water-soluble compound could be prepared. When CoQ 10 was used as the poorly water-soluble compound, CoQ 10 was introduced into the cells and could improve the respiratory ability of the cells. Hepatic disorder could be treated therapeutically in liver disorder model mice. As described above, the obtained lipid structure could be imparted with cell membrane permeability, mitochondrial directivity could be imparted, and the introduced CoQ 10 was functionally effective. ..
実施例８
　本実施例では、難水溶性化合物としてクルクミンを用いた。 Example 8
In this example, curcumin was used as the poorly water-soluble compound.
　ＣｏＱ１０の代わりにクルクミンを用いる以外は、実施例１と同じ調製方法でクルクミン－ＭＩＴＯ－Ｐｏｒｔｅｒを調製した。すなわち、脂質相は以下の組成とし、総流速は５００μＬ／ｍｉｎとし、最終エタノール希釈濃度は、１０％、２０％、３０％または４０％としてマイクロ流路を用いてクルクミン含有脂質ナノ粒子を作製した。
ＤＯＰＥ／ＳＭ／ＤＭＧ－ＰＥＧ　２０００／ＣｏＱ１０／ＳＴＲ－Ｒ８＝９／２／０．３３／２／１．１（モル比） Curcumin-MITO-Porter was prepared by the same preparation method as in Example 1 except that curcumin was used instead of CoQ 10 . That is, curcumin-containing lipid nanoparticles were prepared using a microchannel with the lipid phase having the following composition, a total flow rate of 500 μL / min, and final ethanol dilution concentrations of 10%, 20%, 30%, or 40%. ..
DOPE / SM / DMG-PEG 2000 / CoQ10 / STR-R8 = 9/2 / 0.33 / 2 / 1.1 (molar ratio)
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
　作製した粒子は、実施例１に記載の通り、透析を行い、クルクミン含有脂質ナノ粒子を含む溶液をクルクミン－ＭＩＴＯ－Ｐｏｒｔｅｒとして得た。 The prepared particles were dialyzed as described in Example 1 to obtain a solution containing curcumin-containing lipid nanoparticles as curcumin-MITO-Porter.
　透析前後で得られた溶液の色を確認した。クルクミンは黄色であり、視覚的にクルクミンの溶液への残留を確認することができる。ナノ粒子外のクルクミンは透析による溶液置換によって除去されるために、透析後は、ナノ粒子内のみにクルクミンが残留すると考えられる。透析後に、黄色が確認された場合には、当該黄色は、ナノ粒子内に取り込まれたクルクミンを反映し、黄色の濃淡により、ナノ粒子内に取り込まれたクルクミン量を推定することができる。 The color of the solution obtained before and after dialysis was confirmed. Curcumin is yellow and the residue of curcumin in the solution can be visually confirmed. Since curcumin outside the nanoparticles is removed by solution replacement by dialysis, it is considered that curcumin remains only in the nanoparticles after dialysis. When yellow color is confirmed after dialysis, the yellow color reflects curcumin incorporated in the nanoparticles, and the amount of curcumin incorporated in the nanoparticles can be estimated from the shade of yellow.
　マイクロ流路デバイス上でのエタノール最終希釈濃度を１０％、２０％、３０％、および４０％とし、クルクミン含有脂質ナノ粒子を作製して、透析前後で溶液の色を確認した。その結果、エタノール最終希釈濃度１０％では黄色の着色はとても薄く、２０％以上ではその着色は濃くなる傾向が認められた。 The final dilution concentration of ethanol on the microchannel device was set to 10%, 20%, 30%, and 40%, curcumin-containing lipid nanoparticles were prepared, and the color of the solution was confirmed before and after dialysis. As a result, it was observed that the yellow coloring tended to be very light at the final ethanol dilution concentration of 10%, and the coloring tended to be dark at 20% or more.
　次に、ＤＬＳ法によって得られたクルクミン含有脂質ナノ粒子の粒径とＰＤＩを測定した。結果は図１９に示される通りであった。図１９に示されるように、透析後において、エタノール最終希釈濃度３０％および４０％において、特に良好なＰＤＩを有するクルクミン含有脂質ナノ粒子が得られ、エタノール最終希釈濃度４０％ではＰＤＩが０．３より小さかった。また、粒径は、エタノール最終希釈濃度３０％および４０％において、１００ｎｍ～１５０ｎｍのナノ粒子が得られた。 Next, the particle size and PDI of the curcumin-containing lipid nanoparticles obtained by the DLS method were measured. The results were as shown in FIG. As shown in FIG. 19, after dialysis, curcumin-containing lipid nanoparticles having particularly good PDI were obtained at final ethanol dilutions of 30% and 40%, with a PDI of 0.3 at a final ethanol dilution of 40%. It was smaller. Further, nanoparticles having a particle size of 100 nm to 150 nm were obtained at final ethanol dilution concentrations of 30% and 40%.
　この結果から、本発明の方法において、リン脂質と脂質修飾非電荷親水性ポリマーを含む脂質膜構造体は、ＣｏＱ１０のみならず、クルクミンなどの難水溶性化合物の内包に遊離に用いることができることが明らかとなった。
　図１６で示されるように、リン脂質と脂質修飾非電荷親水性ポリマーを含む脂質膜構造体は、非中空の脂質ナノ粒子であり、粒子内部に脂質膜構造を有する。そして、難水溶性化合物は、脂質ナノ粒子内部の脂質膜構造に取り込まれていると考えられる。 From this result, in the method of the present invention, the lipid membrane structure containing a phospholipid and a lipid-modified uncharged hydrophilic polymer can be freely used not only for CoQ 10 but also for inclusion of a poorly water-soluble compound such as curcumin. Became clear.
As shown in FIG. 16, the lipid membrane structure containing phospholipids and lipid-modified uncharged hydrophilic polymers is non-hollow lipid nanoparticles and has a lipid membrane structure inside the particles. Then, it is considered that the poorly water-soluble compound is incorporated into the lipid membrane structure inside the lipid nanoparticles.
　本発明は、難水溶性化合物の脂質ナノ粒子の製造に関連する分野において有用である。

  The present invention is useful in the field related to the production of lipid nanoparticles of poorly water-soluble compounds.

Claims (24)

  1. 難水溶性化合物を含有し、動的光散乱（ＤＬＳ）法で測定した平均粒子径が６０ｎｍ以下である脂質膜構造体を分散質として分散媒中に含有する分散体であって、前記脂質膜構造体の脂質膜は、リン脂質及び脂質修飾ポリエチレングリコールを含有する、前記分散体。 A dispersion containing a poorly water-soluble compound and having a lipid film structure having an average particle size of 60 nm or less measured by a dynamic light scattering (DLS) method as a dispersoid in a dispersion medium, wherein the lipid film is contained. The lipid membrane of the structure is the dispersion containing phospholipids and lipid-modified polyethylene glycol.
  2. 前記リン脂質は、ジオレイルホスファチジルエタノールアミンとホスファチジン酸及び／又はスフィンゴミエリンである、請求項１に記載の分散体。 The dispersion according to claim 1, wherein the phospholipid is diorail phosphatidylethanolamine, phosphatidic acid and / or sphingomyelin.
  3. 前記難水溶性化合物がＢＣＳ（Ｂｉｏｐｈａｒｍａｃｅｕｔｉｃｓ　Ｃｌａｓｓｉｆｉｃａｔｉｏｎ　Ｓｙｓｔｅｍ）クラス４に属する化合物である、請求項１又は２に記載の分散体。 The dispersion according to claim 1 or 2, wherein the poorly water-soluble compound is a compound belonging to BCS (Biopharmaceutics Classification System) class 4.
  4. 前記難水溶性化合物がＣｏＱ１０である、請求項１又は２に記載の分散体。 The dispersion according to claim 1 or 2, wherein the poorly water-soluble compound is CoQ 10 .
  5. 前記分散媒が水系溶媒である、請求項１～４のいずれかに記載の分散体。 The dispersion according to any one of claims 1 to 4, wherein the dispersion medium is an aqueous solvent.
  6. 前記分散媒はアルコールを含有しない、請求項１～５のいずれかに記載の分散体。 The dispersion according to any one of claims 1 to 5, wherein the dispersion medium does not contain alcohol.
  7. 前記脂質膜構造体の脂質膜は、膜透過性ペプチドをさらに含有する、請求項１～６のいずれかに記載の分散体。 The dispersion according to any one of claims 1 to 6, wherein the lipid membrane of the lipid membrane structure further contains a membrane-permeable peptide.
  8. 前記膜透過性ペプチドが、連続した４～２０個のアルギニン残基からなるポリアルギニンペプチドである、請求項７に記載の分散体。 The dispersion according to claim 7, wherein the membrane-permeable peptide is a polyarginine peptide consisting of 4 to 20 consecutive arginine residues.
  9. ＤＬＳ法で測定した脂質膜構造体の多分散性指数（ＰＤＩ）は、０．３以下である、請求項１～８のいずれかに記載の分散体。 The dispersion according to any one of claims 1 to 8, wherein the polydispersity index (PDI) of the lipid membrane structure measured by the DLS method is 0.3 or less.
  10. 脂質膜構造体のゼータ電位は、１５～２５ｍＶの範囲である、請求項１～９のいずれかに記載の分散体。 The dispersion according to any one of claims 1 to 9, wherein the zeta potential of the lipid membrane structure is in the range of 15 to 25 mV.
  11. 前記分散体は、前記難水溶性化合物を細胞のミトコンドリアに移送するために用いられる、請求項１～１０のいずれかに記載の分散体。 The dispersion according to any one of claims 1 to 10, wherein the dispersion is used for transferring the poorly water-soluble compound to the mitochondria of cells.
  12. リン脂質、膜透過性ペプチド、脂質修飾ポリエチレングリコール並びに難水溶性化合物を溶解したアルコール溶液と水系溶媒とをマイクロ流路構造体のマイクロ流路の入口に連続的に供給し、マイクロ流路内で前記アルコール溶液を水系溶媒で希釈して、難水溶性化合物を含有する脂質膜構造体を分散質として含有する分散体をマイクロ流路の出口から回収する工程を含む、分散体の製造方法。 An alcohol solution in which a phospholipid, a membrane-permeable peptide, a lipid-modified polyethylene glycol, and a poorly water-soluble compound are dissolved and an aqueous solvent are continuously supplied to the inlet of the microchannel of the microchannel structure in the microchannel. A method for producing a dispersion, which comprises a step of diluting the alcohol solution with an aqueous solvent and recovering a dispersion containing a lipid film structure containing a poorly water-soluble compound as a dispersoid from an outlet of a microchannel.
  13. 前記リン脂質が、ジオレイルホスファチジルエタノールアミンとホスファチジン酸及び／又はスフィンゴミエリンであり、前記膜透過性ペプチドが、連続した４～２０個のアルギニン残基からなるポリアルギニンペプチドである、請求項１２に記載の製造方法。 12. The phospholipid is dioleyl phosphatidylethanolamine and phosphatidic acid and / or sphingomyelin, and the membrane-permeable peptide is a polyarginine peptide consisting of 4 to 20 consecutive arginine residues. The manufacturing method described.
  14. 前記脂質膜構造体は、リン脂質、膜透過性ペプチド、及び脂質修飾ポリエチレングリコールを含有する、請求項１２又は１３に記載の製造方法。 The production method according to claim 12 or 13, wherein the lipid membrane structure contains a phospholipid, a membrane-permeable peptide, and a lipid-modified polyethylene glycol.
  15. マイクロ流路へのアルコール溶液及び水系溶媒の各供給量を、マイクロ流路の出口から回収される分散体のアルコール濃度が４０％以下になる量に制御する、請求項１２～１４のいずれかに記載の製造方法。 According to any one of claims 12 to 14, the supply amounts of the alcohol solution and the aqueous solvent to the microchannel are controlled so that the alcohol concentration of the dispersion recovered from the outlet of the microchannel is 40% or less. The manufacturing method described.
  16. マイクロ流路の出口から回収した分散体からアルコールを除去する工程をさらに含む請求項１２～１５のいずれかに記載の製造方法。 The production method according to any one of claims 12 to 15, further comprising a step of removing alcohol from the dispersion recovered from the outlet of the microchannel.
  17. アルコールを除去した分散体を濃縮する工程をさらに含む、請求項１２～１６のいずれかに記載の製造方法。 The production method according to any one of claims 12 to 16, further comprising a step of concentrating the dispersion from which alcohol has been removed.
  18. 各工程を０～３０℃の範囲の温度で実施する、請求項１２～１７のいずれかに記載の製造方法。 The production method according to any one of claims 12 to 17, wherein each step is carried out at a temperature in the range of 0 to 30 ° C.
  19. マイクロ流路構造体は、その上流側において、互いに独立した、第１の流動体を導入する第１導入路と、第２の流動体を導入する第２導入路とが、それぞれ一定長を有して合流し、その下流側に向かって１つの希釈流路を形成しており、前記希釈流路は、少なくともその一部において二次元的に屈曲した流路部位を有し、当該屈曲した流路部位は、これより上流の希釈流路の軸線方向ないしその延長方向をＸ方向と、このＸ方向と垂直に交差する希釈流路の幅方向をＹ方向とし、これより上流の希釈流路の流路幅をｙ０とした場合に、Ｙ方向において対向する希釈流路の両側壁面より交互に、流路中心側に向かって、略Ｙ方向（略＋Ｙ方向、略－Ｙ方向）に、１／２ｙ０以上１ｙ０未満の一定高さｈ１、ｈ２．．．を有し、かつＸ方向に一定幅ｘ１、ｘ２．．．を有して突出し、希釈流路の流路幅を規制する構造子が、一定間隔ｄ１、ｄ２．．．をもって少なくとも２つ以上設けられていることで形成されている流路構造体であり、アルコール溶液を第１導入路に導入し、水系溶媒を第２導入路に導入する、請求項１２～１８のいずれかに記載の製造方法。 On the upstream side of the microchannel structure, the first introduction path for introducing the first fluid and the second introduction path for introducing the second fluid, which are independent of each other, each have a constant length. Then, they merge to form one dilution flow path toward the downstream side thereof, and the dilution flow path has a two-dimensionally bent flow path portion at least in a part thereof, and the bent flow path is formed. As for the road portion, the axial direction of the dilution flow path upstream from this or the extension direction thereof is the X direction, and the width direction of the dilution flow path perpendicular to the X direction is the Y direction. When the flow path width is y0, 1 / in the substantially Y direction (approximately + Y direction, approximately −Y direction), alternately from both side walls of the dilution channels facing each other in the Y direction, toward the center side of the channel. Constant heights h1 and h2 that are 2y0 or more and less than 1y0. .. .. And has a constant width x1 and x2 in the X direction. .. .. The structure that regulates the flow path width of the dilution flow path is formed at regular intervals d1, d2. .. .. 12 to 18, wherein the flow path structure is formed by providing at least two of them, and the alcohol solution is introduced into the first introduction path and the aqueous solvent is introduced into the second introduction path. The manufacturing method according to any one.
  20. 　請求項１～１１のいずれか一項に記載の分散体であって、
    　ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、脂質修飾ポリエチレングリコールとを含む、脂質膜構造体を含み、
    　脂質膜構造体は、ユビキノンまたはそのミトコンドリアの内外膜間の生合成経路における前駆体を含む、分散体。     The dispersion according to any one of claims 1 to 11.
    Includes a lipid membrane structure comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleyl phosphatidylethanolamine, and lipid-modified polyethylene glycol.
    A lipid membrane structure is a dispersion that contains precursors in the biosynthetic pathway between the inner and outer membranes of ubiquinone or its mitochondria.
  21. 　請求項１～１１のいずれか一項に記載の分散体であって、
    　ホスファチジン酸及びスフィンゴミエリンからなる群から選択される１以上のリン脂質と、ジオレイルホスファチジルエタノールアミンと、脂質修飾ポリエチレングリコールとを含む、脂質膜構造体を含み、
    　脂質膜構造体は、クルクミンを含む、分散体。     The dispersion according to any one of claims 1 to 11.
    Includes a lipid membrane structure comprising one or more phospholipids selected from the group consisting of phosphatidic acid and sphingomyelin, dioleyl phosphatidylethanolamine, and lipid-modified polyethylene glycol.
    The lipid membrane structure is a dispersion containing curcumin.
  22. 　請求項２０または２１に記載の分散体であって、
    　脂質膜構造体は、ＤＬＳ法による平均粒径が２０～１５０ｎｍであり、多分散性指数は０．３以下である、分散体。     The dispersion according to claim 20 or 21.
    The lipid membrane structure is a dispersion having an average particle size of 20 to 150 nm by the DLS method and a polydispersity index of 0.3 or less.
  23. 　請求項２０～２２のいずれか一項に記載の分散体であって、
    　脂質膜構造体が、膜透過性ペプチドを表出する、分散体。     The dispersion according to any one of claims 20 to 22.
    A dispersion in which the lipid membrane structure represents a membrane-permeable peptide.
  24. 　請求項２３に記載の分散体であって、
    　膜透過性ペプチドが、オクタアルギニン（Ｒ８）およびＳ２ペプチドからなる群から選択されるペプチドである、分散体。     The dispersion according to claim 23.
    A dispersion in which the membrane-permeable peptide is a peptide selected from the group consisting of octaarginine (R8) and S2 peptides.
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US11904006B2 (en) 2019-12-11 2024-02-20 University Of Iowa Research Foundation Poly(diaminosulfide) particle-based vaccine
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